CHAPTER 1 MACROMOLECULAR X-RAY CRYSTALLOGRAPHY 1.1 PROTEIN STRUCTURE DETERMINATION The cause of most human diseases, like cancer, malaria, Parkinson and Alzheimer's, are proteins. As one of the basic components of a cell, proteins are responsible for regulatory mechanisms, defense systems and structural supports. Consequently, in the factory of living cells, proteins are the workers, performing a variety of biological tasks. Each protein has a particular three dimensional structure that dictates its function. Thus, complete understanding of a protein structure will unveil its particular function and hand in hand this relationship between structure and function will give us a valid picture to design new drugs that can be potential therapeutics for treatment of diseases. Even with high-throughput approach, known as structural proteomics, elucidation of the three-dimensional structure of all natural proteins is likely to be a physically impossible task. Recently, X-ray crystallography and Nuclear Magnetic Resonance (NMR) are widely used as the two primary and powerful techniques for protein structure determination at atomic details. X-ray crystallography appears to overcome protein size restriction, a disadvantage of NMR. Although at least 40,000 protein structures have been solved, this number is currently a small fraction of the thousands of proteins that remain to be understood in detail. 1   1.2 PROTEIN CRYSTALLOGRAPHY Crystallography originated as the science of the study of macroscopic crystal forms. It was originally developed to study minerals and subsequently extended to crystals. With the advent of the X-ray diffraction, the science has become primarily concerned with the study of atomic arrangements in crystalline materials, and the definition of a crystal was defined by Buerger (1956) as: “a region of matter within which atoms are arranged in a three-dimensional translationally periodic pattern”. Furthermore, crystallization is one of the several means (including nonspecific aggregation/precipitation) by which a metastable supersaturated solution can reach a stable low energy state by reduction of solute concentration (Weber, 1991). The three stages of crystallization include: nucleation, growth, and cessation of growth. 1.2.1 X-ray crystallography for proteins Before the development of X-ray diffraction, the study of crystals was primarily based on external geometry. In 1912, crystal structure determination was formulated by an experiment of Max von Laue, who produced an interference pattern on a photographic plate when X-rays were passed through a crystalline sample. The obtained diffraction pattern is due to the scattering of X-rays by the electrons in the sample. With the recent development of computer and diffraction equipment, crystal structure determination has become a relatively easy process. Interestingly, X-ray crystallography has become a primary method for structure determination of biological macromolecules, such as proteins. 2   1.3 BASIC CONCEPTS IN CRYSTALLOGRAPHY 1.3.1 Unit cell and lattices A crystal (Fig. 1.1) consists of a large number of molecules, which are arranged in a particular manner. Figure 1.1. A single protein crystal (adapted from Mathias Klod) A regular pattern of arrangement of an array of points periodically in three dimensional spaces is known as a lattice. In a crystal, a unique volume of space, which is repeated in three dimensions, is called a unit-cell. If each box is represented by a point, then the arrangement of all unit-cells will form a lattice. Even though every crystal has a reduced unit–cell (minimum volume), in some crystals we select a bigger unit-cell (that would include two or more smaller unit-cells), which would satisfy the full symmetrical needs of the crystal. The least volume unit-cell in all crystal systems is called the primitive unit-cell and the bigger unit-cell in some selected crystal systems is called a centered unit-cell. The geometry of a unit-cell is defined by three non-coplanar axes (a, b, c) and their inter-axial angles (α, β, γ). A crystal system is named after the symmetrical requirements of that system and it adopts the corresponding unit-cell. The seven systems are triclinic, monoclinic, orthorhombic, tetragonal, trigonal, hexagonal, and cubic. While all other systems use 3   the corresponding unit-cell, the trigonal system uses either a hexagonal unit-cell or a rhombohedral unit-cell, depending on the symmetry of that particular crystal. These seven crystal systems, depending on the use of the corresponding unit-cell, produce 14 Bravais lattices. Figure 1.2. 1.3.2 Unit-cells and 14 Bravais lattices Symmetry, point group and space group Symmetry in a crystal can be defined as the arrangement of atoms occupying minimum volume, identical and repeats itself throughout the crystal. There are three types of basic symmetry operations in crystallography: rotation, reflection and inversion. The rotational symmetry needs an axis to act upon and produces identical images of an object, around the axis. The number of images generated in crystallography by the rotational symmetry can be 1, 2, 3, 4 or 6. The reflection symmetry acts upon a plane and inversion occurs through a point. The 32 point 4   groups in crystallography describe the unique combinations of these symmetry elements (without any translational component applied to them) in a unit-cell. When a translational component is applied to the symmetry operations rotation and reflection, two additional types of symmetry, the screw axis and the glide plane, are generated. The complete description of a crystal, including the crystal system, lattice type and symmetry elements, is known as the space group of that crystal. There are 230 space 4 groups in crystallography and proteins crystallize only in 65 space groups (without inversion and reflection) that do not warrant the need for D amino acids. 1.3.3 Crystals and X-rays Visible light has the advantage of being focused by a lens and thus it can produce an enlarged image of an object. X-rays, on the other hand cannot be focused. However, the electrons in a crystal diffract X-rays and virtually we want to look at the electron distribution. The objective in X-ray crystallography is to grow crystals to an optimum size and quality for diffraction study. Crystals are generally grown to 0.10.3mm by using different techniques. Crystals for small molecules are easier to form than for proteins. This is due to the complexity of protein molecules and their low availability. Protein crystals are grown by several techniques, including the most common vapor diffusion (hanging drop and sitting drop method) and batch methods. 1.3.4 X-ray diffraction X-ray diffraction is best explained if the radiation is taken as a wave, Eq. 1.1. E=Asinωt (Eq. 1.1) where A is the amplitude, ω is circular velocity, t is time and E is the energy of the wave. X-rays interact with matter and get scattered in all directions. These 5   scattered rays travel different lengths as they originate from different places in a crystal. They differ from one another with respect to their phase and amplitude. Two waves interact constructively when they are in phase (their amplitudes are magnified as the sum of the two waves) while the resultant wave decreases in amplitude if the waves are out of phase (Fig. 1.3). Figure 1.3. 1.3.5 The two types of interference Bragg’s law In crystals, atoms diffract X-rays. Each reflection is the combined effect of waves diffracted by all atoms in the crystal, governed by a set of parallel and equally spaced planes that slice all unit-cells in that particular orientation. According to Bragg’s law when X-rays with a wavelength λ are incident on a set of planes with Miller indices hkl (where h, k, l are the integral divisions of the unit-cell axes a, b, c, respectively) and interplanar spacing of dhkl at an angle θ, they will produce a diffracted beam only if θ meets the following condition, Eq.1.2. 2 dhkl sinθ = nλ (Eq. 1.2) where n is an integer (Rhodes, 2000). 6   1.3.6 Reciprocal lattice and Ewald sphere A set of parallel planes with Miller indices hkl in real space is related to a point (hkl) in the reciprocal space. The direction of the reciprocal vector corresponds to the plane normal and the magnitude of the reciprocal vector is equal to the reciprocal of the interplanar spacing of the real space planes. 1.3.6.1 Ewald sphere Bragg’s law can be rearranged in the reciprocal space using Eq. 1.4. sinθ = λ/2dhkl = (1/dhkl) / (2/λ) (Eq. 1.4) The aim of the Ewald sphere is to determine which set of real space planes (represented by the grid points on the reciprocal space) will result in a diffracted signal for a given wavelength, λ, of incident radiation (Fig. 1.4). Figure 1.4. 1.3.7 Ewald Sphere and radius equation Fourirer transform, structure factor and phase problem The diffraction pattern of atoms in a crystal is related to the atomic arrangement through their Fourier transforms. Thus the electron density at any point in the unit-cell can be calculated by Eq. 1.5. ρ (x, y, z) =1/V ΣΣΣ Fhkl e-2πi (hx+ky+lz) (Eq. 1.5) 7   Through this equation, we transform the diffraction effect in inverse space to real space electron density at every point x, y, z. In the above equation, if the structure factor Fhkl is known, we can calculate the atomic positions and thus the real structure. However, in crystallography to calculate the structure factor Fhkl, we need to know the atomic positions. The reverse Fourier transform of the structure factor equation, will give back the atomic position which is our ultimate aim, i.e. to ascertain the location of every atom in the structure from their diffraction pattern. In order to calculate the position of an atom in a structure we need to know two parameters about a diffracted wave: amplitude and the phase. While the amplitude is calculated from the intensity of a reflection, the phase of the wave, which depends on the positions of all atoms with respect to the origin of the unit-cell, is not measurable. This non-availability of phases is called the ‘phase problem’ in X- raycrystallography. 1.4 DATA COLLECTION For crystal structure determination, the intensities of most, if not all, diffracted beams must be measured. All corresponding reciprocal points must be brought to diffracting position by rotating a crystal. First, the geometry of diffraction which includes the shape, size and symmetry information, is confirmed. This is followed by the measurement of intensities which is ultimately related to the distribution of diffracting electrons in a unit-cell. The X-ray diffractometer (Fig. 1.5) consists two parts, the mechanical part to rotate the crystal and the detector to measure the intensities of diffracted beams. There are three independent axes (ω, χ and φ) through which a crystal can be rotated to bring a desired set of planes into a diffracting orientation. Different physical devices, like photographic film, image plate and charged coupled device (CCD) are used to record X-ray reflections. 8   Figure 1.5. X-ray diffractometer 1.5 STRUCTURE DETERMINATION 1.5.1 Phasing techniques Four techniques are commonly used to derive phase information for structure determination. These methods provide a rough estimate of phases initially which is further improved using repetitive model building and refinement. 1.5.1.1 Direct method This method can be used to solve structures containing 100 or less amino acids. It is based on the assumption that the structure is made of similarly shaped atoms and that there is a statistical relationship between sets of structural factors. The other requirement in case of direct method is the requirement for a very high resolution of data, at the order of 1.2Å or better. 1.5.1.2 Molecular replacement This method is generally preferred to solve the phase problem when a good model for a reasonably large fraction of the structure exists. This means that the sequence similarity between the subject protein and the model must be at least 40% with the model being fairly complete in size. This method is very useful when the structures of structurally homologous proteins are to be solved. 9   1.5.1.3 Multiwavelengh isomorphous replacement Developed in the early 1940s, this method makes use of heavy atoms like gold, mercury or platinum. An initial diffraction pattern of a native crystal is collected, followed by soaking the crystal in two or more heavy atom solutions separately and collection of additional data sets. These heavy atoms contain more electrons than normal protein atoms and hence they produce a significantly varying intensity for every corresponding reflection. Therefore besides serving as spot markers, the change in spot intensities of these atoms help calculate initial phases which are further refined over successive refinement cycle. The reason for use of more than one heavy atom is in the fact that different metals bind to different regions in the protein, thereby aiding very much to resolve phase ambiguity. 1.5.1.4 Anomalous dispersion This method degenerates into single wavelength anomalous dispersion (SAD) or multi wavelength anomalous dispersion (MAD), with the latter being the common method used to study protein structures. When X-rays are incident on molecules heavier than carbon, nitrogen or oxygen part of the energy is absorbed and re-emitted at the same wavelength but at a different phase. This scattering is called ‘anomalous’ scattering. Certain atoms produce substantial anomalous scattering when compared toothers in the useful wavelength range. The most common atoms utilized in X-ray crystallography are sulphur for SAD or selenium which replaces the sulphur in the methionine of a protein in MAD. The advantage of this method lies in the requirement for only one single good quality and well diffracting seleno-methionine crystal. 10   1.5.2 Model building and refinement After scaling and indexing a data set using a program like HKL2000 andsolving the phase problem by one of the above methods, an initial rough model of the structure is built. There are several model building programs like O or Coot. Once the initial model is built, the structure is further refined such that the atomic data is bestfitted. Large numbers of systematic and random errors have an effect on the accuracy of the initial model. Refinement is the process of adjusting the model to find a closer agreement between the calculated and observed structure factors by leastsquares methods or molecular dynamics. This refinement is carried out several times until an accurate model of the structure is obtained. 1.5.2.1 R Factor R-Factor or residual factor is the measure of agreement between the model and actual X-ray data. The R factor is given by Eq. 1.6. (Eq. 1.6) where Fobs is the measured structure facture and Fcalc is the structure factor obtained from the model. Usually, the R factor ranges between 0.2 - 0.25 for a good protein structure. 1.5.3 Validation and presentation The structure is refined several times until a sufficiently low and acceptable R factor without affecting other parameters is achieved. The final structure requires 11   validation before it can be presented. There are two important parameters that must be verified. 1.5.3.1 Ramachandran Plot This powerful validation parameter is not used during the refinement process, but is used to check for the stereochemistry of a structure. For good validation, residues in the disallowed region should be further refined to get at least ninety percentage of all the residues in the allowed region. 1.5.3.2 Folding profile methods A potential protein fold is assigned to the subject protein crystal by searching databases for proteins with similar fold. Often proteins with similar sequence identity tend to show a similar fold. This method was established by Eisenberg and coworkers. The refined coordinates (positions of the atoms) are orthogonalized (arranged with respect to three orthogonal axes), even if the unit-cell has nonorthogonal axes. Also, the temperature factor is a good indicator about the thermal vibration of an atom. The solved structure is deposited at the Protein Data Bank (PDB). 12   CHAPTER 2 BIOLOGY BACKGROUND For my Master thesis, I carried out two projects. In the first part, I have obtained the refolding of Brk, an important kinase involved in lymphoma and then initiated crystallization of the refolded protein. In the second part, in collaboration with Prof. Suresh Subramani’s Lab (University of California, San Diego, USA) I have successfully overexpressed GST-Pex8 and GST-Pex20 (two important peroxins in Pichia pastoris) and initiated their crystallization. 2.1 BREAST TUMOR KINASE (BRK) 2.1.1 Protein tyrosine kinase in signal transduction Protein tyrosine kinases (PTKs) are a wide variety of multigene family evolved to perform functions that regulate a range of cellular processes, including cell growth, differentiation, apotosis, motility, adhesion, and cell-to-cell communication (Pawson, 1994). Although phosphorylation of tyrosine residues in target proteins is essential for maintaining cellular homeostasis, this post-translational modification still provides a number of cellular oncogenes, deregulates various signaling pathways and induces transformation. PTKs are therefore important targets for both basic research and drug development efforts (Levizki et al., 1995). The PTK superfamily can be divided into two groups according to the presence of transmembrane and extracellular domains, which enable PTKs possessing them to recognize extracellular ligands, in particular, various peptide growth factors. Specific ligands and intracellular signaling pathways induced by them have been 13   identified for many, albeit not for all, membrane-spanning PTKs (Schlessinger, 2000). PTKs lacking the transmembrane and extracellular sequences are referred to as nonreceptor or non-transmembrane PTKs. Thirty-two genes encoding for non-receptor PTKs clustered into 10 families are present in the human genome (Robinson, et al., 2000, Manning et al., 2002). Activation of the PTK domain of either class of PTK enzymes results in the interaction of the protein with other signal transducing molecules and propagation of the signal along a specific signal transduction pathway (van der Geer et al., 1994). Dysfunction of cellular phosphorylation is associated with several of human diseases, including cancer to diabetes. Each PTK possesses a functional kinase domain that is capable of catalyzing the transfer of the γ-phosphate group of ATP to the hydroxyl groups of specific tyrosine residues in a protein. Although phosphotransfer reactions that are catalyzed by various PTKs are similar with regard to their basic mechanisms, the recognition of substrates by PTKs and, therefore, subsets of proteins phosphorylated by them show a considerable degree of specificity. Abnormal kinase activity has been shown in a variety of human diseases, in particular those involving inflammatory or proliferative responses, such as cancer. Nowadays, more than 400 human diseases have been connected to protein kinases. The ability to modulate kinase activity therefore represents an attractive therapeutic strategy for the treatment of human illnesses. However, despite a wealth of potential targets, only a few kinases are targeted by drug on the market. Analysis of PTK expression in malignant cells, in general or in lymphomas, in particular will lead to a better understanding of oncogenesis, which in turn will lead to novel therapies based on selective inhibition of these PTKs which are identified as involved in malignant transformation. 14   2.1.2 Brk family non-receptor tyrosine kinases Breast tumor kinase (Brk) belongs to a novel family of intracellular soluble tyrosine kinases and is distinct from the Src family kinases (Serfas and Tyner, 2003). This family is derived from the Src tyrosine kinase family tree early in evolution. Although these kinases are thought to be critical for the normal regulation of many biological mechanisms including cell growth, proliferation, differentiation and metabolism in cancer, their physiological functions remain largely unknown. The Brk family of non-receptor PTKs has four members: Brk, Frk, Srms, and Src42A (Serfas and Tyner, 2003). They are defined by a highly conserved exon-intron structure that is quite different from other major intracellular tyrosine kinase families including c-Src (Lee et al., 1998). They have been cloned independently from human, mouse, and rat cells by several laboratories. Brk is also known as PTK6 and Sik (Mitchell, et al., 1994;Vasioukhin, et al., 1995), whereas Frk is also known as Rak, Bsk, Iyk, and Gtk (Cance et al., 1994; Oberg-Welsh and Welsh, 1995; Sunitha and Avigan, 1996; Thuveson et al., 1995). Srms have been cloned and studied only in mice, but its ortholog is present in the human genome, as well (Kohmura et al., 1994). Src42A, also known as Dsrc41, has been cloned and studied only in Drosophila, and it shares 61% amino acid identity with its putative mammalian ortholog Frk (Serfas and Tyner, 2003; Shishido et al., 1991). Brk and Frk are expressed specifically in epithelial cells, intestinal tract, and their expression is upregulated in some epithelial tumors. In contrast, Srms expression is ubiquitous, although found most abundantly in lung, liver, spleen, kidney and testis (Kohmura, et al., 1994). Src42A is expressed in a wide range of tissues during embryonic development. The Brk-family PTKs are highly homologous to the Src-family PTKs, even more so than are the Csk-family PTKs (Robinson, et al., 2000). Their domain 15   structure including three highly conserved domains followed by an SH3 domain, an SH2 domain, and a tyrosine kinase domain is very similar to that of the Src-family PTKs. The SH2 and SH3 domains are reported to have interactions with phosphorylated tyrosine residues and proline rich sequences of target proteins, respectively (Songyang, et al., 1993, Cohen, 1995). Like the Src family PTKs, these domains are involved in both intermolecular associations that regulate signaling cascades, and intramolecular associations that autoregulate protein kinase activity (Sicheri and Kuriyan, 1997;Thomas and Brugge, 1997;Xu, et al., 1997). However, most Brk-family PTKs lack the N-myristoylation site, the main difference from the Src kinases and are thus not specifically targeted to membrane. The only exception from this rule is rodent Frk, which retains the glycine residue in position 2 and is consequently myristoylated and localized to membrane (Sunitha and Avigan, 1996). Subcellular localization of Brk and Frk has been reported (Cance, et al., 1994; Derry, 2000; Haegebarth, et al., 2004). Frk, Brk and Src42A, unlike Srms, possess tyrosine residues near their C-termini, which might negatively regulate these PTKs in a Srclike fashion. Frk and Src42A have been shown to be phosphorylated by Csk and dCsk respectively (Cance, et al., 1994;Read, et al., 2004). 2.1.3 Brk The intracellular tyrosine kinase Brk was cloned from a human metastatic breast tumor (Mitchell, et al., 1994; Qiu H and Miller WT., 2004). Brk (also known as PTK6) shares 80% amino acid sequence identity to Sik, a non-receptor tyrosine kinase in the mouse intestinal epithelial cell (Vasioukhin, et al., 1995). Both Brk and Sik are members of the Frk family of tyrosine kinase and distantly related to Srckinases (Serfas MS. and Tyner AL., 2003; Kasprzycka et al., 2006). While Brk 16   expression is restricted to normal mammary epithelial cells of skin, melanocytes, gastrointestinal tract and prostate, its highest level is expressed in breast carcinomas, melanomas, colon carcinomas, T-cell lymphoma and in various types of squamous cell carcinomas (Vasioukhin et al., 1995; Derry et al., 2003; Petro et al., 2004; Kasprzycka et al., 2006). Interestingly, Brk expression is developmentally regulated. It is detected late in gestation in mouse, at mouse embryonic day 15.5 (E 15.5) in the differentiating granular layer of the skin and at E 18.5 in the differentiating intestine (Vasioukhin, et al., 1995). Brk expression is initiated as cells migrate away from the proliferative zone and begin the process of terminal differentiation. Overexpression of Brk in mouse keratinocytes resulted in increased expression of the differentiation marker filaggrin during calcium-induced differentiation (Vasioukhin and Tyner, 1997). Brk is expressed in many breast carcinoma cell lines and primary breast tumors, but has not been detected in normal human breast tissue (Barker, et al., 1997;Mitchell, et al., 1994), or at any stage of mammary gland differentiation in the mouse (Llor, et al., 1999). Modest increases in Brk levels have been detected in colon tumors and Brk expression increases during the differentiation of Caco-2 colon adenocarcinoma cells (Llor et al., 1999). In prostate cancers, although the expression of Brk is not significantly elevated, its localization is altered from the nucleus of normal cells to cytoplasmic with gradual progression of tumors (Derry et al., 2003). While Brk is distinctly related to Src (structurally with the SH3-SH2-YP motif, and the regulatory C-terminus) its amino acid sequence does not contain an NH2-terminal myristoylation signal that localizes Src to cell membrane, and therefore is not specifically targeted to membrane (Fig. 1) (Vasioukhin, et al., 1995). In fact, its intracellular localization is flexible and can be present in the nucleus as well as the cytoplasm or at membrane (Haegebarth, et al., 2004). The Src homology 3 (SH3) 17   domain plays an important role in intramolecular interactions that regulate kinase activity, interactions with substrates, cellular localization, and association with other protein targets (Pawson, 1995; Qiu and Miller, 2004). The SH3 domain binds the proline-rich sequences, with the consensus PXXP motif, in substrate proteins or interact with polyproline linker region between the SH2 and kinase domain (Sichery and Kuriyan, 1997). The SH2 domain, on the other hand, is essential in controlling interactions. It recognizes and binds to phosphorylated tyrosine residues, with the specificity being determined by the 3-5 amino acids following these tyrosine residues (Songyang et al., 1993). Like the Src family members, the SH3 and SH2 domains of Brk are also involved in intramolecular interactions with the kinase domain to form an autoinhibited conformation (Qiu and Miller, 2002). Brk activity is down-regulated by phosphorylation of its C-terminal tyrosine residue, Tyr-447 in mouse, similar to that of the Src-family PTKs (Fig. 2.1). However, it remains to be determined how this tyrosine becomes phosphorylated in Brk, since it is phosphorylated neither by Brk itself nor by Csk (Qiu and Miller, 2002). Csk is playing this role for the Src-family PTKs (Liu, et al., 1993). Phosphorylation of Tyr-447 in Brk causes an intramolecular interaction of this tyrosine with the SH2 domain, which leads to the binding of the SH3 domain to the linker region connecting the SH2 domain and the tyrosine kinase domain. However, the question is how tightly this phosphorylation site is associated with the SH2 domain (Qiu and Miller, 2004). Recently, a study has reported that this SH2 domain of Brk is not more functionally similar than most SH2 domains and does not show a high affinity for the proposed autoinhibitory tyrosine of Brk (Y447) (Hong et al., 2004; Julie et al., 2007). In fact, the intramolecular interactions in Brk prevent the binding of ATP to critical catalytic residues, rendering its inactive conformation. 18   Mutation of the carboxy-terminal tyrosine of Brk to phenylalanine (Y447F), which is analogous to Y527 in Src results in increased enzyme activity when overexpressed in epithelial cells, supporting a role for this residue in autoinhibition (Derry et al., 2000; Qiu and Miller, 2002). However, mutation of this regulatory tyrosine resulted in a decrease in the ability of Brk to induce anchorage–independent growth of fibroblasts (Kamalati et al., 1996). Thus, physiological regulation of Brk plays a significant role in kinase activity, localization and cell growth. The SH3-SH2-YK motif presents still remains several unanswered questions relative to the expression, conformation and intramolecular interactions of Brk. Figure 2.1. Structure of Src and Brk tyrosine kinases. Brk shares 44% amino acid sequence identity to Src. Like Src, Brk contains three domains including SH3, SH2 and catalytic domain. The lysine at 295 in Src and at 219 in Brk correlates with the ATP binding site. In contrast to Src, mutation of Tyr-447 to Phe in Brk results in decreasing the ability of Brk to induce anchorage-independent growth. Localization of the Brk tyrosine kinase, similar to Src, has been correlated with its activities and plays an essential role in oncogenesis. Brk is localized in the nucleus of normal luminal prostate epithelia as well as of well-differentiated prostate carcinomas, but mainly in the cytoplasmic in poorly differentiated tumors and more aggressive tumor cell lines (Derry, et al., 2003). Therefore, relocalization of the Brk kinase during the progression of prostate tumor supports its function in maintaining the normal phenotype of prostate epithelial cells which might involve in an unknown 19   signaling pathway. In addition, a correlation between tumorigenicity and the subcellular localization of Brk has also been found in oral squamous cell carcinomas (OSCC) (Petro, et al., 2004). Brk is detected in both the cytosol and nucleus of normal oral epithelium (NOE) and mainly presents in moderately differentiated OSCC cells. Nonetheless, in poorly differentiated OSCC cells, Brk was localized in perinuclear regions, supporting the notion that subcellular localization of this tyrosine kinase plays a role in determining its growth regulating functions and tumorigenesis (Petro et al., 2004). A recent study has also demonstrated the influence of different subcellular localization of Brk in mammalian cells on oncogenic properties. HEK 293 cells, which express Myr-HA-PTK6 and the Brk protein is targeted to the plasma membrane by the Src myristoylation signal (Myr), show increased cell proliferation, antiapoptosis, migration and anchorage-independent growth (Kim and Lee, 2009). However, targeting a variant of Brk (NLS-HA-PTK6) to the nucleus of HEK 293 cells suppresses such oncogenic function. These results suggest that the identification of Brk at plasma membrane could be used as a medical prognostic indicator in the progression of various tumors. Nuclear Brk on the other hand may provide a molecular profiling marker for long-term survival in patients with PTK6-positive cancers. 2.1.4 Role of Brk in lymphoma Although Brk is expressed in several types of normal and cancer cells, its actual role in cell physiology and malignant transformation still remain poorly understood (Kasprzycka et al., 2006). Recently, Brk expression has been documented in normal T-cell-rich PBMCs on their activation with mitogenic or T-cell receptor20   targeting stimuli as well as transformed B lymphocytes (Kasprzycka et al., 2006). Three different types of T-cell lymphoma, CTCL, ALK+, and lymphoblastic has been detected with strong expression of Brk (Kasprzycka et al., 2006). This finding suggests that Brk plays a key role in the physiology of such cells, especially in response to antigens. However, identification of the exact role of Brk in T and, possibly, B lymphocyte as well as conclusion for the comprehensive expression pattern of the kinase in normal and malignant lymphocytes or other immune cells has to be further studied. Interestingly, subcellular localization of Brk as well as its retranslocation in some malignant epithelial cells in vivo contributes to important functions involving in either cell survival or apoptosis and in cell growth, migration and invasion (Kamalati et al, 1996; Derry et al., 2003; Petro et al., 2004; Haegebarth et al, 2005). This is demonstrated in the regulation of signaling by epidermal growth factor (EGF) and serum, via EGFR, erbB3 and possibly other cell-surface receptors to changes in phosphoinsitide 3-kinase, Akt, paxilin and Rac 1 activities and interactions. In the Tcell lymphoma and other lymphoid cells, nuclear localization of Brk is mainly observed and supports to be different from that of cells in which Brk is located in the cytoplasm. It suggests that the nuclear localization of Brk in such cells may be related to the interactions of Brk with a different set of intracellular partners as well as involved in binding with nuclear proteins from the Star family, possibly impacting on cell proliferation (Kasprzycka et al., 2006). In addition, Brk expression also plays a key role in the oncogenicity and pathogenesis of lymphomas. Similar to empty vector-transfected BaF3cells, the kinase-silent K219M mutation fails to effectively compensate for the loss of cytokine (IL-3) or serum-mediated stimuli. Furthermore, this kinase-negative mutant was 21   unable to preserve proliferative and survival characteristics of the BaF3 cells on cytokine and serum withdrawal (Kasprzycka et al., 2006). This result suggested that Brk may have different roles between malignant transformation of lymphoid and mammary cells. Thus, the exact mechanism of Brk and its expression in lymphoma remain to be elucidated. The structural study will establish Brk as a potential therapeutic target and help the patients with long-term breast carcinomas for survival (Kasprzycka et al., 2006). 22   2.2 THE IMPORTANT ROLES OF PEX8 AND PEX20 IN PICHIA PASTORIS 2.2.1 Peroxisome biogenesis and degradation 2.2.1.1 Peroxisomal constituents and its functions Peroxisomes are multifunctional organelles present in nearly all eukaryotic cells. Their diameter ranges from 0.1 to 1.0 µM and they are denser (1.21 – 1.25 gm/cm3) than mitochondria (1.18 gm/cm3) (Subramani, 1993). They are delimited by a single membrane which is impermeable to protons and small metabolites, creating an enzymatically and chemically unique microenvironment within the cell (Dansen , 2000). Peroxisomes contain at least one hydrogen-peroxide (H2O2)-producing oxidase and catalase to decompose the hydrogen peroxide (Lazarow and Fujiki, 1985). Peroxisome functions are often specialized by organism, cell type and environmental milieu. The most widely distributed and well-conserved functions are peroxisomes is the H2O2-based respiration. Other functions include ether lipid (plasmalogen) synthesis and cholesterol synthesis in animals, the glyoxylate cycle in germinating seeds (“glyoxysomes”), photorespiration in leaves, glycolysis in trypanosomes (“glycosomes”), and methanol and/or amine oxidation and assimilation in some yeasts (Fukui and Tanaka, 1979; Tolbert, 1981; Veenhuis et al. 1983; Opperdoes 1987; van den Bosch et al., 1992). In human, there is a wide range of metabolic pathways that is involved with peroxisomes (Fig. 2.2), like ß-oxidation of fatty acids, elimination of hydrogen peroxide, synthesis of plasmalogens and cholesterol. The importance of peroxisomes is underscored by several diseases of peroxisomal dysfunction. This is most clearly demonstrated in the human genetic disorder Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum's disease which are characterized by 23   mental retardation, severe neurologic, hepatic and renal abnormalities, and premature mortality (Lazarow and Moser, 1989). Figure 2.2. Electron micrograph of rat liver. Ultrastructure of rat liver peroxisomes (P), mitochondria (M) and smooth ER (SER). The peroxisomal core composed of crystalline urate oxidase is indicated by arrowheads (Stanley and Paul, 2007). 2.2.1.2 Basic views of Lipid and Protein Import into Peroxisomes Similar to other organelles such as mitochondria and chloroplasts, peroxisome biogenesis also requires at least three conceptually distinct processes: the formation of the lipid bilayer, the insertion of membrane proteins into this bilayer, and the import of soluble proteins across the membrane into the matrix. The peroxisomal membrane contains a phospholipid composition that is distinct from that of other organelles and peroxisome has to import these phospholipids (Lazarow and Fujiki, 1985). Due to the lack of nucleic acids, peroxisomes must import all of their protein content (Fig. 2.3). Proteins destined for the peroxisomal membrane are synthesized on free ribosomes in the cytoplasm (Fujiki, 1984; Imanaka, 1996) and posttranslationally imported into the peroxisome in an ATP-independent manner (Diestelkotter, 1993; Imanaka, 1996). Peroxisomal matrix proteins are also synthesized on free ribosomes and posttranslationally imported, although transport into the matrix is an ATP-requiring process (Lazarow and Fujiki, 1985; Subramani, 1993). 24   A Figure 2.3 B C Peroxisome morphology on different growth media (Pichia pasteris). A. Glucose media; B. Methanol media; C. Oleate media. As growing from glucose media to methanol or oleate, peroxisomes in cells are consequently induced to adapt with surviving environmental change: peroxisomes proliferate and import the necessary metabolic enzymes. Peroxisomal matrix proteins are also synthesized on free ribosomes and posttranslationally imported, although transport into the matrix is an ATP-requiring process (Lazarow and Fujiki, 1985; Subramani, 1993). One curious feature of peroxisomal matrix protein import is that it appears to accommodate folded proteins (Glover JR. et al., 1994; McNew JA, Goodman JM., 1994) and even internally crosslinked proteins and gold particles (Walton et al., 1995), indicating that the translocation apparatus can induce the formation of a large pore in the peroxisomal membrane. 2.2.1.3 Components of the peroxisomal matrix and membrane protein import machinery Like the sorting of proteins to other subcellular compartments, protein targeting to peroxisomes is signal dependent. The peroxisome targeting signal 1 25   (PTS1) and PTS2 signals direct proteins to the peroxisomal matrix, whereas membrane PTS (mPTSs) specify a peroxisomal membrane location (Lazarow, 2003) (Fig. 2.4). These PTSs are recognized by soluble, cytosolic receptors – Pex5p for PTS1 (McCollum et al., 1993; Terlecky et al., 1995), Pex7p and its co-receptor, Pex20p, for PTS2 (Leon et al., 2006; Rehling et al., 1996; Stein et al., 2002; Titorenko et al., 1998; Zhang et al., 1996) and Pex19p (and/or other undefined components) for mPTSs (Jones et al., 2004; Snyder et al., 2000). Following cargo recognition, receptor/cargo complexes are delivered to the peroxisomal membrane for further action. The peroxisome membrane has many peroxins that facilitate the import of matrix and membrane proteins. Two subcomplexes, known as the docking (Pex8p, Pex13p, Pex14p, Pex17p) and RING (Pex2p, Pex10p, Pex12p) subcomplexes, are bridged by Pex8p or another protein, Pex3p, to form a larger complex known as the importomer (Agne et al., 2003; Hazra et al., 2002). Figure 2.4 Targeting signals used by peroxisomal proteins. PTSs are located in the boxes along with the consensus sequences, where applicable and conserved variants are shown below these sequences. In each case, an example of a protein containing the PTS is given (Subramani, 2000). 26   The importomer plays a role in matrix, but not membrane, protein import. The PTS1 and PTS2 receptors and their accessory proteins (e.g. Pex20p) ferry cargo from the cytosol and first interact with the docking subcomplex (Heiland and Erdmann, 2005). The receptor/cargo complexes then either enter the matrix, or are deeply embedded in the peroxisome membrane (Dammai and Subramani, 2001; Leon et al., 2006; Miyata and Fujiki, 2005; Nair et al., 2004). This is followed by cargo release into the peroxisome matrix, export/release of the receptors on the peroxisome membrane (Leon et al., 2006; Miyata and Fujiki, 2005; Platta et al., 2005), followed by dislocation/recycling of the receptors from a peroxisome-associated state to the cytosol (Leon et al., 2006; Miyata and Fujiki, 2005; Platta et al., 2005). Figure 2.5. Membrane protein complexes of the peroxisomal protein import machinery. Peroxin-13 (Pex13), Pex14 and Pex17 are constituents of the docking complex for cycling peroxisomal import receptors. Another protein assembly in the peroxisomal membrane comprises the RING-finger-motif-containing peroxins Pex2, Pex10 and Pex12. This motif is a characteristic element of E3 ubiquitin ligases, and this subcomplex is linked to the docking complex by Pex8, which is peripherally attached to the lumenal side of the peroxisomal membrane (Erdmann and Schliebs, 2005). 27   Mutations in any component of the importomer affect the import of peroxisomal matrix proteins, suggesting that the whole importomer is somehow involved in protein translocation across this membrane (Agne et al., 2003). However, certain transient residents of the peroxisomal matrix, such as Pex5p and Pex20p, become peroxisome-associated and protease protected, even in the absence of the RING subcomplex of the importomer, but their entry into the peroxisome is Pex14pdependent (Leon et al., 2006; Zhang et al., 2006). These data suggest that the docking subcomplex may be the true translocon, at least for these proteins, if not for other matrix cargo as well. The RING subcomplex proteins are required for the export/release of receptors on the peroxisome membrane (Leon et al., 2006; Zhang et al., 2006). The dislocation/recycling of the receptors from the peroxisomes to the cytosol requires the action of a receptor recycling machinery, comprised of an E2-like ubiquitin-conjugating enzyme, Pex4p, two AAA-ATPases, Pex1p and Pex6p, that interact with each other in an ATP-dependent manner, and a peroxisomal membrane protein (Pex15p in S. cerevisiae or PEX26 in mammals), which provides a docking site for Pex6p (Leon et al., 2006; Miyata and Fujiki, 2005; Platta et al., 2005). When this receptor recycling machinery is affected, a peroxisomal RADAR (acronym for Receptor Accumulation and Degradation in the Absence of Recycling) pathway becomes evident (Collins et al., 2000; Kiel et al., 2005a; Koller et al., 1999; Leon et al., 2006; Platta et al., 2004). This involves polyubiquitylation (Fig. 2.6) of peroxisome-membrane-associated Pex5p and Pex20p, most likely by redundant UBCs (Ubc1/Ubc4p/Ubc5p in S. cerevisiae), followed by their degradation by proteasomes (Kiel et al., 2005a; Kiel et al., 2005b; Kragt et al., 2005; Leon et al., 2006; Platta et al., 2004). 28   Figure 2.6. Models for the role of ubiquitylation in receptor recycling (or dislocation) from the peroxisome membrane to the cytosol, and in degradation by the RADAR pathway (Leon et al., 2006). 2.2.2 Peroxisomes and human diseases 2.2.2.1 Peroxisome biogenesis disorders The importance of peroxisomes for human health and normal development is underlined by the existence of several inherited diseases in humans, so called peroxisomal disorders. A defect in a peroxisomal gene can lead to a single enzyme deficiency which might affect one specific peroxisomal function or metabolic pathway. However, when the affected protein is a peroxin, which is involved in the biogenesis and maintenance of peroxisomes, several or all peroxisomal functions can be affected, and peroxisomes can be completely absent. This is the case in peroxisome biogenesis disorders (PBDs) (Braverman et al., 1995; Hannah et al., 2006). PBDs lead to progressive metabolic diseases as well as developmental abnormalities that produce 29   distinct dysmorphic features. Peroxisome biogenesis defects are genetically heterogeneous diseases with an autosomal recessive mode of inheritance. They include the Zellweger syndrome (ZS, also called cerebrohepatorenal syndrome), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD). The clinical pictures of these disorders show similarities, but an important difference is a difference in severity, the clinical course being most severe in ZS and mildest inIRD. Exceptional patients present with a still milder phenotype (Marjo and Valk, 2005). 2.2.2.2 Peroxisomal single protein defects The most common of the single enzyme defects, in which peroxisomes are present but a single enzyme function is deficient, is X-linked adrenoleukodystrophy (XALD). XALD (estimated incidence between 1:40,000 and 1:100,000) is based on mutations in the ALD gene encoding an ATP-binding cassette (ABC) transporter protein of the peroxisomal membrane, which is involved in the import/activation of saturated, unbranched very long chain fatty acids (VLCFA) (Mosser et al. 1993; Netik et al., 1999). Defects or a loss of ALD protein lead to an accumulation of VLCFA, and clinically to progressive demyelination/neurodegeneration in the central nervous system, adrenal insufficiency and death within a few years (Aubourg et al, 1993). 2.2.2.3 Diagnosis and therapy Most understanding in the molecular defects and pathophysiology of the peroxisomal disorders has been made by studying peroxisome biogenesis in yeast mutants and analyzed in vivo by the generation in knock-out mice. Great promise for the early diagnosis of PBDs lies in the molecular analysis of PEX genes, and molecular testing is evolving. Laboratory diagnosis usually involves blood and urine 30   analysis (e. g., plasma VLCFA analysis, analysis of plasmalogens in erythrocytes, αoxidation of phytanic acid) followed by detailed biochemical and morphological studies in patient’s fibroblasts. An alternative approach is the so called pharmacological gene therapy, which uses certain drugs (e. g., 4-phenylbutyrate) to increase the expression of peroxisomal genes which can either complement the function of the disease gene or increase the number and matrix protein content of peroxisomes (Haan et al., 2006). 2.2.3 Pichia pastoris Pex8 and Pex20: role in peroxisomal matrix protein import machinery 2.2.3.1 Pichia pastoris Pex8 (Pex8) Pex8 has been described above as a central organizer of the importomer, a multisubunit complex of peroxisomal membrane and associated proteins, which is essential for matrix protein import into peroxisomes. Interestingly, Pex8 is necessary for peroxisomal matrix import of proteins carrying either a PTS1 or a PTS2 sequence (Lan et al., 2006). To release the PTS2 cargo within the peroxisomes, Pex8p interacts with the cargo-loaded pex20 and pex7 complex. The interaction nature of these three proteins is still unknown. Which protein in this complex can first bind to Pex8 and cotranslocate into the peroxisome or both proteins in cargo complex interact with Pex8p are to be verified. To perform these critical functions, Pex8 must be translocated into the peroxisome matrix. The question is how Pex8 is itself released from Pex7 and Pex20 cargo inside peroxisome. 31   2.2.3.2 Pichia pastoris Pex20p (Pex20) Pex20 has also been related to the PTS2 pathway and cotranslocates with Pex7 and Pex20 into the peroxisome. However, this interaction mechanism and the cycle between the cytosol and the peroxisome as part of an “extended cycle” of this complex are still unknown. Recently, a conserved cysteine residue of this Pichia pastoris Pex20 has been identified to play an essential role for its recycling from peroxisome to the cytosol (Leon et al., 2006). Nevertheless, this residue is not completely necessary for the function of the protein because similar to what happens in the recycling mutants, Pex20 (C8S) is constitutively degraded by the RADAR pathways. It may be relative to the ubiquitylation mechanism but whether or not this cysteine is ubiquitylated remains unknown. Identification of the Pex20 structure as well as the components and interactions mediating the recycling and RADAR pathways will help in the global understanding of peroxisome biogenesis in Pichia pastoris as well as shed light to treat human peroxisomal biogenesis defects. 2.3 OBJECTIVE I have undertaken two independent projects for my research. The 3D structure of the full length wild type Brk protein will provide a good background to understand the tumorigenesis progression cell proliferation and survival. In addition, the combination of this backbone structure and its mutants may be helpful to analyze their significant roles in the pathogenesis research of lymphoma and breast cancer researches, in general. Some preliminary proteomic studies have been performed with a variety of comprehensive expression patterns of Brk and its partner as well as its probable interactions either in vitro or in vivo. However, the exact mechanisms 32   responsible for the Brk-mediated control over cellular functions remains to be elucidated. The solution structure of the SH2 domain has been solved. However, the structure full length Brk only will offer promising answer to how this tyrosine kinase expresses and influences in malignant cell transformation of lymphocytes. Along with some of the interaction studies and substrate binding mechanism, Brk may shed light on some specific signal transductions, which are related to most long-term breast cancer patients. Furthermore, structure determination of this enzyme, coordinated with functional analysis, will help in the development of morphological and molecular markers for cancer therapy. Pex8 plays an important role in peroxisomal matrix protein import mechanism. Without the understanding of the structures of the involved proteins, the whole map of all pathways related to peroxins will not be complete. Interestingly, the interaction between Pex8, Pex20 and Pex7 in the PTS2 path-way in Pichia pastoris still remains a mystery. Hence, each full length structure (Pex8, Pex20 and Pex7) will be useful in addressing questions like which parts of them play a significant role in interaction and what are their actual biological functions in Pichia pastoris. These structures and full understanding of their complexes will be excellent models for elucidating the potential links between the cellular import-deficiency of yeast mutants and corresponding pathways in human cell lines. In turn, potential cure for human peroxisomal biogenesis disorders can be systematically attempted. 33   CHAPTER 3 MATERIALS AND METHODS 3.1 EXPRESSION AND PURIFICATION OF RECOMBINANT WT-BRK 3.1.1 Construction of expression for WT-Brk The humanized cDNA encoding breast tumor kinase gene was synthesized using reverse transcriptase polymerase chain reaction (RT-PCR). This reaction was used with oligo(dT) primers and total mRNA extracted from human breast tumor cells (T47D) as template. A 5μg- 20μl RT reaction with 2μl DTT (0.1 M, Invitrogen), 4μl 5X RT buffer (Invitrogen), 2μl dNTP (10 mM, Roche), RNAase OUTTM (Invitrogen) 1μl, 1μl Oligo dT (50 μg/μl, Invitrogen), 5μl RNA (1 μg / μl), 1μl Superscript3 RT (Invitrogen), 4μl nuclease-free reaction water was used. The RT reaction was followed by a normal PCR reaction to amplify the gene of interest. A 50μl PCR reaction mix with 3μl MgCl2 (25mM, Promega), 5μl Mg free 10X PCR buffer (Promega), 1.5μl dNTP (10 mM, New England Biolab), 2μl each forward and reverse primers (10 μM), 1μl cDNA (2ng), 34.5μl dH2O, 1μl Taq Polymerase (Promega) was used. The PCR reaction was carried out for 35 cycles. The forward and reverse primers BRK14FP (TTCTT CATATG ATG GTG TCC CGG GAC CAG) and BRK14RP (TTCTT GGATCC TCA GGT CGG GTT CTC GTA GCT), respectively, provided WT-Brk gene with the NdeI and BamHI restriction enzyme sites, respectively (underlined). Briefly, the initial denaturation of the PCR cycle was set at 94°C for 4 minutes and followed by 35 cycles of: denaturation at 94 °C, annealing at 55°C and extension at 72 °C, for 30 sec at each step. The final extension was carried out at 72 °C for 10min. PCR products were analyzed by 1% 34   agarose gel electrophoresis and the PCR product at the expected size was cloned to the pGEM T-EZ vector for sequencing and subsequent cloning. 3.1.2 Bacterial expression The WT Brk gene was sub-cloned into a modified pET 14b (Novagen) vector (with His-tag) for expression. The construct was transformed into BL21 (DE3) pLysS competent cells. The BL21 (DE3) pLysS cells that contain the pET 14b: Brk construct, which confer ampicillin and chloramphenicol resistance. Hence the transformants were plated onto an LB agar plate containing 100 μg mL-1 ampicillin and 34 μg mL-1 chloramphenicol for selection. Initially, a series of experiments were carried to check and optimize protein expression. Expression was tested using 50 ml cultures before it was scaled up to higher volumes. The first series of trials involved testing the construct using time based expression experiments. Four 50 ml cultures were inoculated with 2 ml of culture grown overnight. Bacteria were initially grown to an optical density (OD600) of 0.6 (log phase) at 37 °C, and induced with different concentrations of IPTG (0.25, 0.5, 0.75 and 1 mM). Full length WT-Brk showed protein expression but most of the protein formed inclusion body and very less soluble protein. Normally, very low expression temperatures (less than or around 30 °C) and lower IPTG concentration (less than or around 0.1mM) can increase the solubility of expressed protein. Six 1L culture was grown to log phase at 37°C and the temperature was lowered to 16°C before induction with 0.1 mM IPTG. The cultures were grown for 16 hours and then the cells were harvested by spinning them at 5,000g for 20 minutes. 35   3.1.3 Solubilization, purification and refolding from inclusion body 3.1.3.1 Solubilization The inclusion body fraction of WT-Brk is defined as the component that gets pelleted during centrifugation of the cell lysate. The pellet from the previous section was lysed two times by French press in 50mL denaturing buffer A containing 8 M urea, 20 mM Tris-HCl (pH 8.5), 0.5 M NaCl, 100 μM PMSF. The suspension was centrifuged at 40,000g for 30 minutes at 4°C. The supernatant was then applied to the purification step. 3.1.3.2 Purification The supernatant was mixed with 5 ml of the nickel-NTA agarose (Quigen) resin and rotated on a rocker for overnight binding at 4 °C. The resin containing bound WT-Brk protein was subjected to washing with 30 mL of 20 mM Tris-HCl, 0.5 M NaCl (pH 8.5), 100 μM PMSF, 5 mM 2-mercaptoethanol, 1% Tween 20 and 5 mM imidazole. The protein was eluted with 10 mL of buffer A containing 250 mM imidazole as elution buffer. 3.1.3.3 Rapid dilution The eluted supernatant was then refolded by 30 fold rapid dilution in a redox refolding buffer [20 mM Tris-HCl (pH 8.5), 0.5 M NaCl, 0.5 M arginine, 0.5 mM oxidized glutathione and 5 mM reduced glutathione]. The refolding solution was incubated for 24 hours at 4°C with continuous stirring and then dialyzed for 36 hours against 20 mM Tris-HCl (pH 8.5), 0.5 M NaCl without urea. The dialyzed solution was clarified and concentrated to 5ml before being further purified by Fast protein liquid chromatography (FPLC). 36   3.1.3.4 Size-exclusion chromatography The His-tag refolded WT-Brk protein was purified by size exclusion chromatography on a pre-equilibrated Hi-Load 16/60 Superdex-75 column (GE Healthcare) with buffer [20 mM Tris-HCl (pH 8.5), 0.5 M NaCl in an AKTA Explorer 10 system (GE Healthcare) at a flow rate of 0.8 ml/min. Protein peaks were detected by UV absorbance at 280nm. The fractions under the peak were analyzed by SDS-PAGE for purity. The most pure fractions were pooled together and concentrated until 0.5, 1, 1.8, 2, 3 mg/ml, respectively and frozen using liquid nitrogen for long term storage. 3.1.3.5 Western blot analysis for denatured and refolded WT-Brk The denatured and refolded WT-Brk proteins were verified by Western blot analysis using a Brk specific primary antibody. The eluted denatured protein after purified by affinity chromatography and the refolded protein after rapid dilution and purification by size exclusion chromatography, were run on 12% SDS-PAGE with PageRulerTM Prestained Protein Ladder as a standard marker. The SDS-PAGE gel was passively transferred to a PVDF membrane using a Mini Trans-Blot cell (BioRad) following the manufacturer’s instruction. The non-specific sites were blocked by incubating the membrane overnight at 4 °C in PBST buffer (1X PBS containing 0.05% Tween 20) plus 5% skimmed milk. After the incubation, the membrane was washed three times with PBST buffer without skimmed milk for total 30 minutes. Then, a rabbit monoclonal anti-Brk antibody Sigma-Aldrich was used as a primary antibody for binding overnight at 4 °C in PBST containing 5% skimmed milk. The next morning, the primary antibody was removed and the membrane was washed three times with PBST buffer without skimmed milk. The membrane was incubated 37   with a goat anti-rabbit antibody (Sigma-Aldrich) as the secondary antibody. The Brk band was documented by developing the membrane with Pierce ECL Detection Reagent 1and Pierce ECL Detection Reagent 2 (Thermo Scientific) mixture and exposure to X-ray film. 3.1.3.6 Dynamic light scattering A dynamic light scattering (DYNA Pro) study was carried out on the concentrated WT-Brk sample to analyze its polydispersity. 30μl of the protein was aliquoted and microcentrifuged at 20,000g for 30 minutes at 4 °C. 20μl of the protein was used for analysis. The cuvette was checked for any previously bound proteins by measuring the count by taking only distilled water. The count was maintained below 10 before the protein sample was loaded in the cuvette. 3.1.3.7 Circular dichroism spectroscopy Circular dichroism spectra were acquired on a J-810 Spectropolarimeter (Jasco) using a quartz cuvette with 1 mm path length (Hellma). The spectra was averaged over five scans and recorded at the wavelength region from 190 to 280 nm with 0.1 nm resolutions using a scan speed of 50 nm/min and a response time of 8 seconds. For each sample, a different spectrum was obtained by subtracting the sample buffer spectrum from the refolded WT-Brk spectrum. The circular dichroism signal is reported in units of mean residue molar ellipticity which is a quantity normalized to the concentration of protein residues. 38   3.1.3.8 Intrinsic tryptophan fluorescence measurement Fluorescence measurements were carried out using a Perkin-Elmer LS50 Luminescence spectrometer. All measurements were done in a 10mm quartz cuvette. The excitation and emission slits were set at 5nm. The intrinsic tryptophan fluorescence was measured using an excitation wavelength of 295nm to avoid tyrosine emission. Emission spectra were recorded from 300-400nm, using a 5nm band pass for both excitation and emission. The final concentration of Brk used in all measurements was 20μM. For thermal unfolding studies, refolded WT-Brk were boiled and held at each temperature for 5 minutes except for 70 degree that was heated for both 5 minutes and 10 minutes. For chemical denaturing, 2M and 4M urea was added to protein samples and incubated for 1hour. 3.1.3.9 Crystallization Once the polydispersity index of WT-Brk was sufficiently deemed good (in the range 10-30%), the pure protein was spun down at 16,000g for 30 minutes in order to minimize any contamination from any fortuitous substances such as dust particles and fibers. The Sparse matrix Screen1, Screen2 kits from Hampton research and JBScreen 1,2,3,4,5,6,7,8 from Jena BioScience were used to set up for crystallization at protein concentrations of 0.5, 1, 1.8 and 2 mg/ml. Crystallization drops containing 1 μl refolded protein solution were combined with 1 μl of corresponding reservoir solution. Crystallization trials were set up at room temperature at 16°C and 4°C. For microbatch experiments, 24-well plates from Greiner were used. In this case, sitting drops containing 1 μl protein solution were mixed with 1 μl of reservoir solution and covered with 8μl paraffin oil. These experiments were also incubated at room temperature 16°C and 4°C. 39   3.2 EXPRESSION AND PURIFICATION OF RECOMBINANT GST-Pex8 AND GST-Pex20 3.2.1 Bacterial expression The pGEX KG - Peroxin factor 8 (GST-Pex8) and pGEX KG - Peroxin factor 20 (GST-Pex20) constructs having a TEV site for proteolytic cleavage were provided by our collaborator Prof. Suresh Subramani (University of California, San Diego). These constructs were transformed into BL21 (DE3) E.coli competent cells for expression. The BL21 (DE3) cells containing the pGEX KG construct which confers ampicillin resistance. Hence the transformants were plated onto an LB agar plate containing 100 μg mL-1 ampicillin for selection. Similar to the WT-Brk expression, first small scale express tests (50 mL) was first made to optimize the conditions. The expression condition was optimized at 1 mM IPTG concentration and 30 °C for 3 hours. The fusion protein was expressed in six 2.8 liter flasks with one liter culture in each. The cultures were grown for 3 hours at 30 degree induction and then the cells were harvested by spinning them at 9,000g for 20 minutes. 3.2.2 Affinity purification of GST-Pex8 and GST-Pex20 After centrifugation, the cell-free extract was appliedd to a glutathione sepharose (GE Healthcare) column with 1:200 (v/v) binding ratio. The fusion protein was eluted by 20 mM reduced glutathione and 20 mM Tris-HCl. The crude extracts, flowthrough, wash, eltuted fractions and pure protein were analysed by 12% SDSPAGE as described by Sambrook et al (Sambrook, J., et al, 1989). 40   3.2.3 Anion exchange chromatography The Hi-Trap Q Sepharose 5 mL anion exchange column was equilibrated with 10 mL column volumes of phosphate buffer without salt [20 mM NaH2PO4 ( pH 7.0) for GST-Pex8 and pH 7.5 for GST-Pex20, filtered] at a flow rate 3 ml/min in an AKTA system. The GST-Pex8 and GST-Pex20 proteins were concentrated to 5 mL and injected into the FPLC system. Each of these proteins was loaded at flow rate no greater than 1 ml/min. Each of these proteins was eluted with a linear gradient of 0 to 1 M NaCl using a computer-controlled gradient mixer. The unbound washed proteins and eluted fractions were detected by 12% SDS-PAGE. 3.2.4 Gel filtration A HiLoad 16/60 Superdex 200 pre-grade column (16 mm diameter and 60 cm bed height) was equilibrated with buffer containing 20 mM NaH2PO4 and 300 mM NaCl (pH 7.0 for GST-Pex8 and pH 7.5 for GST-Pex20). The Superdex 200 was connected to an AKTA explorer 10 system (GE Healthcare, USA). 5 mL of affinity purified GST-Pex 8 and GST-Pex 20 proteins was injected separately into the loop of this system. These proteins were eluted at a flow rate 0.5 ml/min and 0.8 ml/min, respectively. Protein peaks were detected by UV absorbance at 280nm. All the fractions were collected and analyzed by 12% SDS-PAGE before further tests, such as Dynamic Light Scattering (DLS) for dispersal characteristics and peptide mass finger printing for protein identity verification. 3.2.5 Mass spectroscopy identity for GST-Pex8 and GST-Pex20 proteins Protein identification by peptide mass fingerprinting and post-source decay MS/MS analysis was carried out using an ABI Voyager-DE™ STR Biospectrometry™ Workstation and Micromass Q-tof Tandem Mass Spectrometer. 41   In-gel digestion to produce peptides for analysis by mass spectrometry was carried out by following the protocol from Protein and Proteomic Centre (PPC), Department of Biological Sciences, National University of Singapore using trypsin (modified and sequencing grade, Promega). Coomassie-stained protein bands were destained prior to in-gel digestion using the method from PPC. Purification and concentration of peptides were isolated from in-gel digestion of protein bands by Zip-tip (method from Advanced Protein Technology Centre, Hospital for Sick Children, Toronto, Canada). Database searches were carried out on in-house Mascot server that was licensed to Umea University of Matrixscience (www.matrixscience.com) using the current version of the NCBI website. 3.2.6 Protein concentration identification All the protein concentrations were determined by the Bradford assay using bovine serum albumin as a standard. The Protein Assay Dye Reagent concentrate (Bio-Rad) was mixed with diluted protein samples and added to the cuvette (1 cm light path). The solution in cuvette was mixed well and incubated at room temperature for 5 minutes prior to reading absorbance at 595 nm. 42   CHAPTER 4 RESULTS AND DISCUSSION 4.1 WT-BRK REFOLDING, STRUCTURE ANALYSIS AND CRYSTALLIZATION 4.1.1 Bacterial expression The full length (451aa) recombinant human construct WT-Brk gene was cloned into the pET14b vector and was over-expressed as a His-tagged protein in E.coli BL21 (DE3) pLysS cells. The level of protein expression was verified by 12% SDS-PAGE with Coomassie staining (Fig. 4.1). The protein migrated to a position consistent with the expected size of 52 KDa. Figure 4.1. Overexpression of Brk. WT-Brk in pLyss cells were induced by the different concentration of IPTG. Lane 1, molecular weight marker. Lane 2, uninduced cell lysate. Lane 3,5,7,9, insoluble fraction after cell lysis after induced at 0.7, 0.8, 0.9 and 1mM IPTG, respectively. Lane 4, 6, 8, 10, soluble fraction after induced by 0.7, 0.8, 0.9 and 1mM IPTG, respectively. 43   Most (95%) of the Brk protein was in inclusion body in our time course experiments (Fig. 4.2). Even in all our low temperatures, low IPTG concentrations and the baculovirus – insect cell expressions, 90% of Brk was insoluble (data not shown). Figure 4.2. WT-Brk was over-expressed in inclusion body after 3 hours induction 4.1.2 WT-Brk affinity purification The insoluble Brk protein was solubilized in 8 M urea and purified using the Nickel-NTA resin (Fig. 4.3). Figure 4.3. Purification of Brk. Brk was purified from inclusion body after solubilizing the insoluble fraction by 8 M urea. Lane M, molecular weight marker. Lane I, insoluble fractions. Lane U, uninduced cell lysates. Lane Fl, flowthrough. Lane W1, W2, W3 indicates washing steps 1, 2, 3, respectively. Lane E(I) shows eluted protein from insoluble fraction. 44   4.1.3 Purification of refolded WT-Brk by gel filtration The Brk protein purified by the previous step was refolded by rapid dilution in 30 volumes of 20 mM Tris, 0.5 M NaCl buffer. The rapid diluted and presumably refolded protein was dialyzed against the same buffer to remove all residual urea and concentrated to 5 mL. The protein was further purified by gel filtration at a flow rate is 0.8 mL/min. Brk eluted out as the 1st peak with some other contaminants, peaks 2 and 3. The fractions containing pure Brk were pooled out after SDS-PAGE analysis and concentrated using Amicon microcentrifuge device (Fig. 4.4). The final concentration of refolded Brk could be up to 3 mg/mL with minimum aggregation. This pure concentrated protein (Fig. 4.5) was used for further biophysical a experiments. Figure 4.4. b Size-exclusion chromatography profile of refolded WT- Brk. (a) Purification profile of refolded Brk and (b) SDS-PAGE analysis under the peak with (lanes 2 to 4, peak 1; lanes 5 to 8, peak 2; lanes 9 to 12, peak 3). 45   4.1.4 The homogeneity of refolded Brk Dynamic Light Scattering (DLS) has been successfully applied in the study of unfolding-refolding reactions of protein molecules (Damaschun et al., 1991; Klaus et Figure 4.5. The homogenecity of refolded Brk. (a) Pure refolded Brk. (b) Coomassie Blue staining of SDS-PAGE in native condition. Based on the positive control, the refolded Brk protein shows only one single band as indicated by the arrow. al., 1992). Furthermore, protein aggregation is commonly observed during protein refolding and this phenomenon could be investigated by the intermolecular interactions from DLS data analysis. Interestingly, although the shape and density of the solute particles in biological macromolecules are typically unknown, with the help of DLS, the molecular weight can also be estimated by measuring the value of diffusion coefficient (DT). More importantly, crystallization is the main step in X-ray crystallographic studies. This process, which involves co-operative, step-wise addition of identical and monodisperse molecules to a growing ordered assembly, is very much limited by a different and non-specific precipitation from heterogeneous aggregate. Many useful data about sample polydispersity of proteins can be provided by this technique as the intensity of scattered light is proportional to the square of the mass of the solute particle (Adrian, 1994). 46   In our experiment (Figs. 4.6, 4.7), we elucidated the homogenous characteristic of purified and refolded Brk. As a result of some aggregation during refolding, the yield of Wt-Brk protein decreased significantly after dialysis. Experimentally, refolded Brk can be harvested at 3 mg/mL with minimum aggregation. Hence, it seems to be the end point of protein concentration for setting up crystallization. Figure 4.6. The dispersity characteristic of refolded Brk using Dynamic Light Scattering (DLS). Protein concentration is 2 mg/mL. The results show homogeneity without any aggregation. Red color vertical frame indicates the average radius of the molecule. Red color horizontal frame indicates the molecular weight and polydispersity index, which indicates that a sample is monodisperse if the index is less than 20%, medium disperse for 20% to 30% and mainly polydisperse if the index is more than 30%. 47   Figure 4.7. DLS profile for refolded Brk at 3 mg/mL concentration. The red vertical frame shows that the radius of the sample is increased, due to some aggregation. Thus, the maximum protein concentration for crystallization can be only 3 mg/mL for refolded WT-Brk. 4.1.5 Characterization of refolded Brk by Western blot It is very important to confirm the purity of this protein at each step of refolding. A series of western blotting experiments was performed in order to confirm the identity of the refolded protein as well as the yield of this protein after renaturation. The expected size of this protein was verified. However, the concentration as well as the yield of the soluble refolded decreased as a result of precipitation during the rapid dilution and dialysis steps. The 52 KDa WT-Brk protein was detected and recognized by specific rabbit anti-Brk primary antibody (from Sigma) and goat anti-rabbit IgG secondary antibody, Fig 4.8. 48   Figure 4.8. Western blot analysis of Brk refolding. The protein is located at the correct size (52 KDa). Lane 1 shows pre-stained molecular marker. Lane 2 indicates the band of cell lysate after solubilizing insoluble Brk with 8 M urea. Lane 3 shows the the presence of refolded Brk after purification by FPLC and lane 4 shows the higher yield of denatured protein after purification by an affinity column which indicates the loss of protein during rapid dilution and dialysis. 4.1.6 Circular Dichroism (CD) of WT-Brk    The process of refolding of an insoluble protein is a highly complex process. Several intermediates are formed during this process. For in vitro protein unfolding and refolding studies using denaturants such as urea or guanidine hydrochloride, the protein conformation can be consecutively monitored through intrinsic fluorescence emission or circular dichroism techniques. In this experiment, using Far UV circular dichroism measurements, we try to see whether the secondary structures of Brk are formed properly during the unfolding and refolding processes. The ellipticity of Brk remained almost constant at all wavelengths at 8 M urea (when the protein is 49   denatured completely), Figs. 4.9 and 4.10. However, the spectrum showed the two characteristic absorptions at 210 and 220 nm for a mixture of α-helices and β-strands for the refolded and purified (by gel filtration) protein. It could be concluded that the protein did not lose its secondary structures during the renaturation step without any strong denaturants. In addition, CD spectroscopy can be used to study the conformational stability of a protein under stress, such as thermal stability or pH stability. The temperature scanning is one of the simple, easy and efficient techniques which estimate the conformation and packing characteristics of a refolded protein. Many proteins aggregate or precipitate quickly after they are unfolded ("melted"), making unfolding irreversible. The reversibility of the unfolding reaction can be assessed by cooling the sample and then heating again to see if the unfolding reaction is duplicated. If the protein precipitates or aggregates as it is unfolded, the melting reaction will be irreversible, and the melting temperature will reflect the kinetics of aggregation and the solubility of the unfolded form of the molecule as well as the intrinsic conformational stability. The cooperativity of the unfolding reaction is measured qualitatively by the width and shape of the unfolding transition. A highly cooperative unfolding reaction indicates that a protein exists initially as a compact, well-folded structure, while a very gradual, non-cooperative melting reaction indicates that the protein exists initially as a very flexible, partially unfolded or as a heterogeneous population of folded structures. Initially, a single wavelength at which major changes in the conformation of secondary structures occurred was chosen to monitor the structural feature of WT-Brk. Herein, the absorption of this spectrum was observed to be much altered and modified at 209 nm. Based on this wavelength, we have continuously recorded the CD signals of the refolded protein as the temperature was 50   raised from 20 to 90 ºC. The results demonstrate that the structure of refolded Brk is well-packed and completely denatured at 70 ºC, Fig. 4.11.. Figure 4.9. A far-UV CD spectrum for refolded WT-Brk. Unfolded WT-Brk in 8M urea 1 hour (blue) and refolded WT-Brk (red line), which shows the presence of α-helices and β-strands. The protein concentration is 20 μM. Figure 4.10. The CD-spectra of WT-Brk at different temperatures. The blue curve is for 20 ºC and the red curve is for 70 ºC. Note the major conformational and structural differences at the wavelength of 209 nm. 51   Figure 4.11. Thermal denaturation scanning for refolded WT-Brk. 4.1.7 Intrinsic tryptophan fluorescence spectroscopy for refolded Brk The content and state of burial or exposure of the three aromatic amino acid residues (tryptophan, tyrosine and phenylalanine) in a protein contribute to the intrinsic fluorescence of the protein. Changes in the intrinsic fluorescence can be used to monitor structural changes in proteins. Frequently, in these proteins, spectral shifts are observed as a result of phenomena such as binding, protein-protein interactions and denaturation. The fluorescence of a folded protein is the total of the fluorescence from individual aromatic residues. Protein fluorescence is generally excited at 280 nm or at longer wavelengths, usually at 295nm. In fact, most of the emissions are due to the excitation of tryptophan residues with very few emissions from tyrosine and phenylalanine. It is observed that tryptophan (Trp) has much stronger fluorescence and higher quantum than the other two aromatic residues. The intensity, quantum yield and wavelength of maximum fluorescence of tryptophan are dependent on solvent or environment. In other words, only tryptophan has an absorbance maximum near at 295 nm and a maximal emission will be more sensitive to the polarity of the environment. In case of free tryptophan dissolved in aqueous solvent or a solvent52   exposed Trp in a particular protein, it is hypothesized that the spectra shows up the emission maximum at 350 nm wavelength. On the other hand, a Trp dissolved in an organic solvent or buried deep inside the protein like α-lactalbumin and lysozyme will have an emission maximum near 320 nm. To check whether WT-Brk is well-folded, we measured intrinsic tryptophan fluorescence at different thermal-denaturing and chemical unfolding conditions (high concentration urea). The results experimentally demonstrate that most tryptophan residues, as hydrophobic residues, are buried in the folded protein and show increased intensity as they become exposed when the protein is unfolded. This observation is consistent with some recent studies on the intramolecular interactions of Brk. Lee and Kim (2005) have recently shown that Trp-184 of PTK6 plays an important role in autophosphoryaltion (at Tyr-447) and phosphorylation of other substrates, like Sam68. Herein, this tryptophan residue is important for the interaction of the linker that contains Trp-184 with the kinase domain. In turn, this interaction is absolutely required for catalytic activity of PTK6. However, Sam 68 is reported not to be phosphorylated by the W184A mutant of Brk. 53   Figure 4.12 The intrinsic tryptophan fluorescence changes of WT- Brk when thermally unfolded. Tris buffer was used as a negative control (blue line near the X-axis). During this experiment, refolded Brk (red) showed fluorescence at intensity 200 (at 350 nm). Its tryptophans would have tried to bury themselves. With 5 minutes thermal denaturation at 45 ºC (green), the fluorescence of unfolded WT-Brk has increased the maximum as all tryptophans are presumably exposed. Note that the fluorescence decreases as the time and temperatures are increased. One possible reason might be the aggregation of the unfolded protein. The chart represents the average of three runs. Therefore, the ability of WT-Brk to refold can be verified through tryptophandependent fluorescence change. WT-Brk was first denatured using temperature and the fluorescence changes were measured, Fig. 4.12. After that, the protein was denatured at two concentrations of urea (4 and 8 M) and the fluorescence changes were measured, Fig. 4.13. The result indicates that tertiary structure of Brk may be properly formed. 54   Figure 4.13. Intrinsic tryptophan fluorescence under chemical stress with urea (2 M and 4 M). The refolded Brk expose more tryptophan residues by indicating more fluorescent intensities after treated protein with high concentrations of urea. The fluorescent intensity of refolded protein is lightly decreased after denatured by 4M urea due to the precipitation of protein. 4.1.8 Crystallization of refolded Brk Along with several biophysical experiments like CD spectra, DLS and Intrinsic tryptophan fluorescence spectroscopy have been carried out that aim to verify whether protein refolded properly, some preliminary crystallization trials is studied. Approximately more than 200 crystallization conditions for screening have been tried. However, the refolded protein has not till been crystallized. One possible reason for this failure is the low amount of refolded Brk. For total 6 litters of cultures, 55   we can harvest only 0.75 mg in 250 μL of buffer (the maximum concentration can get at 3 mg/mL). This amount may not be enough for more crystallization screening experiments. 4.2 PEROXISOME PROTEIN EXPRESSION, PURIFICATION AND CRYSTALLIZATION 4.2.1 Bacterial expression and affinity purification for GST-Pex8 (peroxin factor 8) and GST-Pex20 (peroxin factor 20) constructs The construct pGEX-KG: peroxisome factor 8 (and 20 in a separate construct) with TEV cleavage site was kidly provided by our collaborator Prof. Subramani. Each of these constructs was transformed into BL21 (DE3) E. coli cells for protein overexpression. The recombinant protein solubility prediction package from University of Oklahoma predicted 61.4% chance of solubility for GST-Pex20 when expressed in E. coli and 58.4% for GST-Pex8. After suitable small scale expression, 6 L cultures were grown. The proteins were expressed as soluble fractions. The GST-Pex8 protein showed two bands (65 and 110 KDa) in 12% SDS-PAGE. The expected size was 110 KDa and the 65 KDa band could be due to cleavage by proteases or non-specific binding of another protein. GST-Pex20 showed only one band at 66 KDa, Fig. 4.14. These proteins were first purified using an affinity column and further purified by gel filtration using a Superdex 200 column. 56   Figure 4.14. Overexpression of GST-Pex20 and GST-Pex8 in BL21 (DE3) E. coli cells. Lane-1 marker; flowthrough of GST-Pex20 and GST-Pex8 are shown in at lanes 2 and 7, respectively. Lanes 4 and 5 are washes for GST-Pex20 and lanes 8 and 9 are for GST-Pex8. Purified GST-Pex20 and GST-Pex8 are shown in lanes 6 and 10 at lane 10. It is observed that the quality of these fusion proteins may not high and enough for further purified, like FPLC. We added 0.01% lysozyme during cell disruption to increase the Pex8 yield, Fig. 4.15. For GST-Pex20 also, we are optimizing protein yield. The final concentration of these proteins is around 1 mg from 1 L culture. a b Figure 4.15. Optimized purification of GST-Pex8 by lysozyme addition via affinity chromatography using GSTrap FF 1 mL from GE Healthcare. a) Lysozyme was added to lysis buffer b) No lysozyme was added to lysis buffer. 57   4.2.2 Gel filtration chromatography for peroxisome proteins Following the initial GST purification, each of these fusion proteins was concentrated until to 100 μL, approximately 4 mg/L (from 1 L culture). The proteins were applied to Superdex 200 (1.6 x 60 cm, Pharmacia) and eluted at a flow rate of 1 ml per minute. The peaks were analyzed by 12% SDS-PAGE and visualized by Coomassie staining, Fig. 4.16. For GST-Pex8, we found that this fusion protein usually aggregated with some small fragments below 30 KDa indicating in the first peak of gel filtration. Unfortunately, the contaminating background signals can still be detected in this part. Interestingly, the full-length Pex8 protein could be isolated in the second peak. To improve the yield of this protein and avoid protein aggregation, we have tried to optimize the cell growth condition, and more starting material. Gel filtration was followed for GST-Pex20 using a similar protocol (data not shown). 4.2.3 Optimized purification for GST-Pex 20 using anion exchange column In this study, because less amounts of these peroxisome proteins were obtained and that too with contamination from other non-specifically bound proteins, we tried to use other techniques to remove the unwanted proteins completely, thereby improving the purity of the GST-Pex8 and GST-Pex20 fusion proteins. Ion exchange chromatography is one of the most important methods for protein purification. This method removes non-specific bands very efficiently. Large volumes of protein solution can be applied to ion exchange columns, often much greater than the volume of the column itself. The subject protein can be eluted even by step gradients, as an alternate to the use of a linear gradient, which is effective if the appropriate concentrations of salt are known, usually from initial linear gradient experiments. 58   a   b   Figure 4.16. Gel filtration profile for GST-Pex8. (a) Gel filtration of GST-Pex8 using a Superdex 200 column. (b) Relatively purer and soluble GST-Pex8 was located at peak 2. Peak 1 shows some aggregated protein at the void volume and Peak 3 indicates low molecular weight contamination. The principle of this technique is fundamentally based on charge-charge interactions between the sample protein and the charges immobilized on the resin of our choice. In this case, our protein is negatively charged in the lysis buffer (pI= 5.5 for GST-Pex8 and pI = 5.0 for GST-Pex20) and can bind to the positively-charged 59   immobilized resin. The column is first equilibrated with a low ionic strength buffer and our proteins are allowed to bind to the resin. The bound proteins, including some non-specifically bound proteins, are eluted using a gradient of increasing ionic strength buffer. The protein of interest, say GST-Pex20, gets eluted in a pure form, at a different ionic strength of the eluting buffer (hence a different time point) than other proteins, Fig. 4.17. Alternatively, the pH of the elution buffer can also be varied to elute the protein of interest. Lowering the pH of the mobile phase buffer causes the bound molecules to become more protonated and hence more positively charged. The result is that the fusion protein no longer has the capability to form a strong ionic interaction with the positively charged solid support which causes the molecule to elute from the chromatography column. We exploited this option for GST-Pex20 which also increased the yield of purified protein. Similar anion exchange chromatography purification for GST-Pex8 is being optimized (This data have not shown). However, we found that the final purity of this application to GST-Pex8 is still not increased. One possible reason is that the charges of GST-Pex8 seem to be more positive in lysis buffer pH 7.5. Thus, we suggest that GST-Pex8 fusion protein can be systematically purified by affinity with lysozyme addition and then only followed by gel filtration purification. 60   a   b    Figure 4.17. Anion exchange purification of GST-Pex20. (a) Chromatograph (b) SDS-PAGE of the fractions. The first peak was of non-specifically bound proteins. The second peak contains mainly pure GST-Pex20. Because the yield of GST-Pex20 fusion protein is much more improved by using anion exchange chromatography, it would be an interesting and efficient way to 61   firstly purify this protein by affinity and secondly followed anion column purification before highly purified by gel filtration. The identity of GST-Pex8 (Fig. 4.18) and GST-Pex20 (Fig. 4.19) was verified by peptide mass finger printing. Figure 4.18. Peptide mass fingerprinting of GST-Pex8. The result confirmed that the sample is Pex8 with a score of 79. Figure 4.19. Peptide mass fingerprinting of GST-Pex20. The result confirmed that the sample is Pex20 with a score of 326. 62   CHAPTER 5 CONCLUSION AND FUTURE STUDIES 5.1 CONCLUSION The solubilization of denatured wild type Breast tumor kinase (Brk or PTK6) was successfully carried out by using rapid dilution refolding and gel filtration even though the amount of this refolded protein have not been enough to optimize crystallization trials. Along with this protein, overexpression and crystallization of GST-Pex8 and GST-Pex20 were also completed. The major aim of the project is to solve the crystal structure of these full-length proteins to address the following questions. How does Brk play a significant role in T-lymphoma as well as other lymphoid cells? How do intraperoxisome Pex8 and cytosolic Pex20 activate outside proteins to translocate into peroxisomal membrane? Which parts of them are associated with Pex7 to transport essential peroxisomal proteins into peroxisome following the PTS2 pathway? While most of the activities of Brk have been eventually reported and published, the structure of the full-length this protein has to be explored and the real mechanism related to the signal transduction pathway of this protein has not been furnished yet. Although the SH2 domain structure for the Brk protein has recently been solved, much more important questions concerning this protein are still to be answered. Although some preliminary crystallization attempts have been failed, we have successfully refolded this wild type kinase and reported important biophysical characteristics. These initial studies on conformational and structural characteristics of refolded WT-Brk are very encouraging for the next optimum crystallization steps. With these promising results, we hope that the crystal structure of full-length WT-Brk 63   will come true soon and shed new lights on diagnosis and treatment therapy for breast cancer. Peroxins are proteins that are required for various aspects of peroxisome biogenesis including assembling peroxisome membrane, importing most of the peroxisomal matrix proteins, peroxisome proliferation and peroxisome inheritance. Two of such peroxins, Pex8 and Pex20 that are involved in the PTS2 pathway, are known to play key roles in transporting important peroxisomal matrix proteins into the matrix of the peroxisome. These proteins also interact with the Pex7 receptor to translocate proteins that have the PTS2 sequence into the peroxisome. Even though most of the activities and functions of these proteins have been predicted, full understanding of these proteins will be possible after theirs structure are known. Once the structures of Pex7, Pex8 and Pex20 are known, the whole map of peroxisome biogenesis and other related peroxisomal matrix inport mechanism will be very clear. In the present study, GST-Pex8 and GST-Pex20 proteins have successfully been overexpressed and purified. These purified proteins may initially use for further studies on biophysical analysis and crystallization strategy. 5.2 FUTURE STUDIES Because the relationship between protein structure and its function plays essential roles in the active sites, protein-ligand interaction in vivo clinical therapies, it is necessary to extract all useful information from the structure and interpret the fnction of the protein. In this project, we have successfully refolded Brk and the identification of its sequence is by Peptide Mass Fingerprinting and N-terminal protein sequencing is underway. Additionally, we are preparing for a potential activity assay for the kinase along with our crystallization attempts. We are also tesing the 64   refolding of Brk with guanidine-HCl denaturation to avoid the formation of urea crystals. It will also be interesting refold the two functionally important mutant variants (K219, an inactivated kinase; Y447F, an activated mutant) with similar refolding conditions. As soon as these three variants structure are solved, it will pave an easier way to analyze their functions and predict their potential influence in lymphoma. Studies of peroxisome and its function in various model organisms, such as plants, mammals and human have revealed their remarkable roles in biogenesis, morphogenesis and evolution. 23 peroxins and their mutants regulate the assembly, division and inheritance of peroxisomes and cause lethal peroxisome biogenesis disorders in humans. The behavior behind these stories is that these peroxisomes usually form complex networks of physical and functional interactions together. No part of these peroxins could be neglected in their control of the precise dynamics of peroxisome biogenesis. Pex8 and Pex20 eventually contribute a major role in importing peroxisomal proteins into the matrix. These two proteins have been successfully expressed in E. coli and solubilized. However, their yields are still quite low. Some conditions are further being optimized. Firstly, the addition of lysozyme (or other chemicals) to lysis buffers will break more cells and increase the amount of soluble proteins. Secondly, the DLS studies on these proteins should be further confirmed. Finally, the ion exchange protocol must be further optimized to improve the yield and purity of the proteins. There may be interesting results if cation exchange and hydrophobic interaction chromatography could be used. Once these proteins are completely ready, promising quality crystals will not be in silence. 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J Cell Biol. 132(3):325-34   74   [...]... refolding of Brk, an important kinase involved in lymphoma and then initiated crystallization of the refolded protein In the second part, in collaboration with Prof Suresh Subramani’s Lab (University of California, San Diego, USA) I have successfully overexpressed GST-Pex8 and GST-Pex20 (two important peroxins in Pichia pastoris) and initiated their crystallization 2.1 BREAST TUMOR KINASE (BRK) 2.1.1... tyrosine kinase Brk was cloned from a human metastatic breast tumor (Mitchell, et al., 1994; Qiu H and Miller WT., 2004) Brk (also known as PTK6) shares 80% amino acid sequence identity to Sik, a non-receptor tyrosine kinase in the mouse intestinal epithelial cell (Vasioukhin, et al., 1995) Both Brk and Sik are members of the Frk family of tyrosine kinase and distantly related to Srckinases (Serfas MS and. .. neurologic, hepatic and renal abnormalities, and premature mortality (Lazarow and Moser, 1989) Figure 2.2 Electron micrograph of rat liver Ultrastructure of rat liver peroxisomes (P), mitochondria (M) and smooth ER (SER) The peroxisomal core composed of crystalline urate oxidase is indicated by arrowheads (Stanley and Paul, 2007) 2.2.1.2 Basic views of Lipid and Protein Import into Peroxisomes Similar... mitochondria and chloroplasts, peroxisome biogenesis also requires at least three conceptually distinct processes: the formation of the lipid bilayer, the insertion of membrane proteins into this bilayer, and the import of soluble proteins across the membrane into the matrix The peroxisomal membrane contains a phospholipid composition that is distinct from that of other organelles and peroxisome has... translocated into the peroxisome matrix The question is how Pex8 is itself released from Pex7 and Pex20 cargo inside peroxisome 31   2.2.3.2 Pichia pastoris Pex20p (Pex20) Pex20 has also been related to the PTS2 pathway and cotranslocates with Pex7 and Pex20 into the peroxisome However, this interaction mechanism and the cycle between the cytosol and the peroxisome as part of an “extended cycle” of this complex... to understand the tumorigenesis progression cell proliferation and survival In addition, the combination of this backbone structure and its mutants may be helpful to analyze their significant roles in the pathogenesis research of lymphoma and breast cancer researches, in general Some preliminary proteomic studies have been performed with a variety of comprehensive expression patterns of Brk and its partner... demonstrated in the regulation of signaling by epidermal growth factor (EGF) and serum, via EGFR, erbB3 and possibly other cell-surface receptors to changes in phosphoinsitide 3 -kinase, Akt, paxilin and Rac 1 activities and interactions In the Tcell lymphoma and other lymphoid cells, nuclear localization of Brk is mainly observed and supports to be different from that of cells in which Brk is located... Analysis of PTK expression in malignant cells, in general or in lymphomas, in particular will lead to a better understanding of oncogenesis, which in turn will lead to novel therapies based on selective inhibition of these PTKs which are identified as involved in malignant transformation 14   2.1.2 Brk family non-receptor tyrosine kinases Breast tumor kinase (Brk) belongs to a novel family of intracellular... capable of catalyzing the transfer of the γ-phosphate group of ATP to the hydroxyl groups of specific tyrosine residues in a protein Although phosphotransfer reactions that are catalyzed by various PTKs are similar with regard to their basic mechanisms, the recognition of substrates by PTKs and, therefore, subsets of proteins phosphorylated by them show a considerable degree of specificity Abnormal kinase. .. and even internally crosslinked proteins and gold particles (Walton et al., 1995), indicating that the translocation apparatus can induce the formation of a large pore in the peroxisomal membrane 2.2.1.3 Components of the peroxisomal matrix and membrane protein import machinery Like the sorting of proteins to other subcellular compartments, protein targeting to peroxisomes is signal dependent The peroxisome ... of Brk, an important kinase involved in lymphoma and then initiated crystallization of the refolded protein In the second part, in collaboration with Prof Suresh Subramani’s Lab (University of. .. overexpressed GST-Pex8 and GST-Pex20 (two important peroxins in Pichia pastoris) and initiated their crystallization 2.1 BREAST TUMOR KINASE (BRK) 2.1.1 Protein tyrosine kinase in signal transduction... three stages of crystallization include: nucleation, growth, and cessation of growth 1.2.1 X-ray crystallography for proteins Before the development of X-ray diffraction, the study of crystals