Chapter 4.3 Results Elucidation of amino acid residues important for sjIPNS solubility Approaches to enhance protein properties such as stability and solubility have been reported for many systems (Lehmann et al., 2000; Sim and Sim, 1999). However, thus far, only mutations that decrease solubility have been reported for IPNS isozymes (Sim and Sim, 1999; Loke and Sim, 1999b). Rarely, perhaps no study has yet been employed to replace amino acid residues for the specific aim of improving the solubility of IPNS enzymes. The low solubility of sjIPNS has fortuitously made it a suitable candidate to challenge the engineering of crucial amino acid residues to improve its solubility. Therefore, the focus of this section is to identify amino acid residues that may be potentially important for the solubility of sjIPNS and to use site-directed mutagenesis to investigate the effects of amino acid replacements on the solubility of sjIPNS. It would be exciting if amino acid substitutions that can increase sjIPNS solubility were elucidated. This would serve as a platform for us to understand how the effects of specific amino acid mutations translate into altered protein expression. The results of this study have shown that although IPNS isozymes are closely related in terms of sequence homology and function, they can be classified into two groups based on their expression profiles at 37ºC and 25ºC (Section 4.1.5). Group I consists of four bacterial IPNS, namely scIPNS, sfIPNS, sIIPNS and nIPNS, which have been shown to be highly soluble when the induction temperature is lowered from 37°C to 25°C. In contrast, sjIPNS, the only member in Group II, is insoluble at both temperatures. It is intriguing that despite the high nucleotide (77%-85%) and amino acid (70%-82%) sequence homology amongst the bacterial isozymes, the solubility of sjIPNS in E. coli appears to be different compared to other bacterial isozymes. This implies that the amino acid sequence of each isozyme, especially in nonhomologous regions, may dictate their folding and expression characteristics. Consequently, we propose that the low solubility nature of sjIPNS, uniquely different from the rest of the compared IPNS isozymes, is due to the non-conserved amino acid sequences between them. More specifically, it is anticipated that unique differences in the amino acid residues in sjIPNS Chapter Results relative to the other four soluble isozymes within the non-conserved regions may be the determinants for low solubility of sjIPNS. Hence, we hypothesize that by replacing certain amino acid residues at these determinant sites in sjIPNS with conserved amino acid residues naturally occurring at the corresponding sites in all the soluble bacterial isozymes may improve sjIPNS solubility. To test our hypothesis, comparative sequence analysis followed by rational amino acid replacements were performed. 4.3.1 Comparative amino acid sequence analysis of sjIPNS and other soluble bacterial IPNS isozymes In order to identify relevant amino acid residues for site-directed mutagenesis, the amino acid sequences of the four soluble bacterial IPNS enzymes from Group I were selected as representative soluble bacterial IPNS sequences and used in our sequence comparison with sjIPNS. This was carried out using CLUSTAL W Multiple Sequence Alignment Program (Version 1.6) (Thompson et al., 1994). Fig. 4.17 shows the alignment between sjIPNS (sequence depicted in blue) and the soluble isozymes (sequences depicted in black). The conserved residues between the compared IPNS enzymes were highlighted in gray using the BOXSHADE program (Version 3.2). In addition, functionally important conserved residues shown by IPNS crystal structures and series of mutagenic experiments (reviewed in Section 2.5.4) involved in binding Fe2+ ion and ACV substrate in IPNS were highlighted by “*”. Our focus is on the non-conserved regions between sjIPNS and the soluble isozymes, which correspond to the non-highlighted regions in the alignment (Fig. 4.17). Next, was to further elucidate sites within these regions that satisfied our defined criterions, which are sites whereby the amino acid residues in the soluble bacterial IPNS are wholly conserved either in identities or in a particular amino acid property (i.e. hydrophobic, polar uncharged, charged acidic and charged basic; colored in pink, yellow, blue and green respectively in the alignment) but are absent in sjIPNS. Based on these criterions, eight sites with conserved amino acid Chapter Results identities occurring only in the soluble IPNS isozymes and not in sjIPNS were identified (marked by “♦”). These correspond to Ser19, Ala37, Asp112, Glu120, Ile156, Ala202, Arg242 and His260 of sjIPNS. In addition, three more sites whereby the soluble IPNS isozymes showed a consistent preference for a particular amino acid property over that found in sjIPNS were also identified (marked by “•”). These correspond to Tyr160, Thr308 and Thr309 of sjIPNS. At these three positions in all the soluble IPNS, hydrophobic amino acid residues as oppose to the polar uncharged residues in sjIPNS are found. Altogether, eleven sites scattered throughout the primary amino acid sequence of sjIPNS were elucidated. Information on these sites was tabulated in Table 4.3. 4.3.2 Proposition of sites for site-directed mutagenesis to investigate its influence on sjIPNS solubility Eleven amino acid positions were identified whereby the soluble scIPNS, sfIPNS, slIPNS and nIPNS differ from the insoluble sjIPNS. Intuitively, these elucidated sites may cause sjIPNS to be expressed in the insoluble aggregates. Upon close examination, the soluble IPNS enzymes have residues with higher hydrophobicity present at seven out of the eleven identified sites (sjIPNS position 19, 120, 160, 242, 260, 308 and 309) as compared to sjIPNS (Table 4.3). Particularly, at site 160, 308 and 309, a prevalent conservation of hydrophobic property is observed in the soluble IPNS proteins. Take site 309 for example, leucine, valine and isoleucine hydrophobic residues are found at this position in the various soluble IPNS (Table 4.3), indicating that hydrophobicity is preferred at this site in all the soluble IPNS. However, polar threonine is found at this site in sjIPNS. Next, we pose the question of whether replacing the amino acid residues at these identified sites in sjIPNS with those found in the soluble IPNS isozymes can improve the solubility of sjIPNS. To address this possibility, site-directed mutagenesis was used to construct sjIPNS single mutants for all the eleven sites. The choices of amino acid changes are Chapter Results Fig. 4.17 Amino acid sequence alignment of insoluble sjIPNS and soluble bacterial IPNS isozymes (scIPNS, sfIPNS, slIPNS and nIPNS). Numbering of amino acid positions highlighted is with respect to sjIPNS. 37 19 scIPNS sfIPNS slIPNS nIPNS sjIPNS 1 1 ♦ ♦ MPVLMPSAHVPTIDISPLFGTDAAAKKRVAEEIHGACRGSGFFYATNHGV MPILMPSADVPTIDISPLFGDDPDAKTHVAQQINKACRGSGFFYASHHGI MPIRMPSAHVPTIDISPLFGTDPDAKAHVARQINEACRGSGFFYASHHGI --MKMPSAEVPTIDVSPLFGDDAQEKVRVGQEINKACRGSGFFYAANHGV MPILMPSAEVPTIDISPLSGDDAKAKQRVAQEINKAARGSGFFYASNHGV scIPNS sfIPNS slIPNS nIPNS sjIPNS 51 51 51 49 51 ∗ DVQQLQDVVNEFHGAMTDQEKHDLAIHAYNPDNPHVRNGYYKAVPGRKAV DVQQLQDVVNEFHGTMTDEEKYDLAINAYNSANPRVRNGYYMAVEGKKAV DVRRLQDVVNEFHRTMTDQEKHDLAIHAYNENNSHVRNGYYMARPGRKTV DVQRLQDVVNEFHRTMSPQEKYDLAIHAYNKNNSHVRNGYYMAIEGKKAV DVQLLQDVVNEFHRNMSDQEKHDLAINAYNKDNPHVRNGYYKAIKGKKAV 112 scIPNS sfIPNS slIPNS nIPNS sjIPNS 101 101 101 99 101 156 scIPNS sfIPNS slIPNS nIPNS sjIPNS 120 ♦ ♦ ESFCYLNPDFGEDHPMIAAGTPMHEVNLWPDEERHPRFRPFCEGYYRQML ESWCYLNPSFGEDHPMIRSGTPMHEVNIWPDEKRHERFRPFCEQYYRDMF ESWCYLNPSFGEDHPMIKAGTPMHEVNVWPDEERHPDFRSFGEQYYREVF ESFCYLNPSFSEDHPEIKAGTPMHEVNSWPDEEKHPSFRPFCEEYYWTMH ESFCYLNPSFSDDHPMIKSETPMHEVNLWPDEEKHPRFRPFCEDYYRQLL 160 151 151 151 149 151 ♦ • ∗ KLSTVLMRGLALALGRPEHFFDAALAEQDSLSSVSLIRYPYLEEYPP--V QLSKTLMRGFALALGKPEDFFDANLPEDDTLSAVSLIRYPHLKAYPP--V RLSKVLLRGFALALGKPEEFFENEVTEEDTLSAVSMIRYPYLDPYPEAAI RLSKVLMRGFALALGKDERFFEPELKEADTLSSVSLIRYPYLEDYPP--V RLSTVIMRGYALALGRREDFFDEALAEADTLSSVSLIRYPYLEEYPP--V 199 199 201 197 199 ∗ ♦ ∗ ∗ ∗ ♦ KTGPDGQLLSFEDHLDVSMITVLFQTQVQNLQVETVDGWRDIPTSENDFL KTGPDGTKLSFEDHLDVSVITVLFQTEVQNLQVETVNGWQDLPTSGDDFL KTGPDGTRLSFEDHLDVSMITVLFQTEVQNLQVETVDGWQSLPTSGENFL KTGPDGEKLSFEDHFDVSMITVLYQTQVQNLQVETVDGWRDLPTSDTDFL KTGADGTKLSFEDHLDVSMITVLYQTEVQNLQVETVDGWQDIPRSDEDFL 202 scIPNS sfIPNS slIPNS nIPNS sjIPNS 242 260 scIPNS sfIPNS slIPNS nIPNS sjIPNS 249 249 251 247 249 ♦ ∗ ∗ ∗ ∗ ∗ VNCGTYMAHVTNDYFPAPNHRVKFVNAERLSLPFFLNGGHEAVIEPFVPE VNCGTYMGYLTNDYFPAPNHRVKFINAERLSLPFFLHAGHTTLMEPFSPE INCGTYLGYLTNDYFPAPNHRVKYVNAERLSLPFFLHAGQNSVMKPFHPE VNAGTYLGHLTNDYFPSPLHRVKFVNAERLSLPFFFHAGQHTLIEPFFPD VNCGTYMGHITHDYFPAPNHRVKFINAERLSLPFFLNAGHNSVIEPFVPE 308 309 scIPNS sfIPNS slIPNS nIPNS sjIPNS 299 299 301 297 299 •• GASEEVR-NEALSYGDYLQHGLRALIVKNGQT DTG-GKELNPPIEYGDYLQHGFHALIAKNGQT DTG-DRKLNPAVTYGEYLQEGFHALIAKNGQT GAPEGKQGNEAVRYGDYLNHGLHSLIVKNGQT GAAGTVK-NPTTSYGEYLQHGLRALIVKNGQT Hydrophilic, acidic : D,E Polar, uncharged : N, Q, S, T, W, Y Hydrophilic, basic : H, K, R Hydrophobic : A, C, F, G, I, L, M, P, V 125 Table 4.3 Elucidated sites where the identities or properties of the amino acid residues in sjIPNS are different from that of soluble bacterial IPNS isozymes. Position in sjIPNS 19 37 112 120 156 160 sjIPNS Amino acid residue Ser Ala Asp Glu Ile Tyr Amino acid property Polar, uncharged Hydrophobic Hydrophilic, acidic Hydrophilic, acidic Hydrophobic Polar, uncharged Soluble bacterial IPNS isozymes (scIPNS, sfIPNS, slIPNS & nIPNS) Amino acid residue Amino acid property Phe Hydrophobic Cys Hydrophobic Glu Hydrophilic, acidic Gly Hydrophobic Leu Hydrophobic Phe Hydrophobic sjIPNS single mutant constructed Ser19Phe Ala37Cys Asp112Glu Glu120Gly Ile156Leu Tyr160Phe (sfIPNS, sIIPNS, nIPNS) Leu Hydrophobic Tyr160Leu Hydrophobic Polar, uncharged Polar, uncharged Hydrophobic Ala202Pro* Arg242Thr His260Asn Thr308Ala (scIPNS) 202 242 260 308 Ala Arg His Thr Hydrophobic Hydrophilic, basic Hydrophilic, basic Polar, uncharged Pro Thr Asn Ala (scIPNS, sIIPNS, nIPNS) Pro Hydrophobic - Hydrophobic Thr309Leu Hydrophobic Thr309Val Hydrophobic - (sfIPNS) 309 Thr Polar, uncharged Leu (scIPNS) Val (sIIPNS, nIPNS) Ile (sfIPNS) * Mutant Ala202Pro was constructed previously in our laboratory and the mutant clone was used here for our investigation. Chapter Results guided by the identities of amino acid residues present in the soluble IPNS isozymes (Table 4.3). Only one type of substitution was carried out for all the identified positions except at positions 160 and 309. For position 160, the endogenous tyrosine residue in sjIPNS was changed to phenylalanine and leucine separately to construct two sjIPNS single mutants at this position. Likewise for position 309, the naturally occurring threonine in sjIPNS was substituted with leucine and valine separately. Thus, a total of thirteen sjIPNS single mutants were constructed. The list of sjIPNS single mutants constructed in this study was shown in Table 4.3. Another major cogitation when deciding on the amino acid for replacement is that the changes should not affect the catalytic ability of sjIPNS enzyme adversely. Since all the proposed mutations were located within the non-conserved regions, leaving the functionally important conserved residues (marked by “*”) intact, perturbation of the enzyme activities of sjIPNS single mutants was likely to be minimal. 4.3.3 Construction of sjIPNS single mutants To investigate the effects of the proposed amino acid replacements on the expression of soluble sjIPNS in E. coli, site-directed mutagenesis was carried out followed by expression of the resultant sjIPNS mutant clones in E. coli BL21(DE3) in the following Sections. 4.3.3.1 Site-directed mutagenesis, selection and sequence confirmation of sjIPNS single mutants To probe the influence of the thirteen amino acid substitutions at the eleven proposed sites on the solubility of sjIPNS, the approach adopted is to use site-directed mutagenesis for specific alteration of these sites using QuikChangeTM site-directed mutagenesis kit (Stratagene) Chapter Results that employs the in vitro PCR mutagenesis technique. The detailed experimental procedures involved are described in Section 3.2.8. For each mutation, two primers carrying the intended mutation were designed to anneal to the same sequence on the opposite strands of the plasmid. Mutagenic primer sequences for each mutation were shown in Appendix II. Recombinant pET-SJ plasmid was used as the template for the PCR mutagenesis. During temperature cycling, the mutagenic primer pair would anneal to the same sequence on opposite strands of the sjIPNS gene (Fig. 4.18a) and Pfu polymerase would extend DNA synthesis in opposite directions from the 3’ ends of both primers. As such, the amplified product (~6.3kbp) would have the same size as the plasmid template (Fig. 4.18b). Subsequently, the reaction mixture was incubated with DpnI for three hours. DpnI only recognizes target sequences that are methylated. This selection served to remove the methylated parental pET-SJ plasmid. The final mixture was then transformed into E. coli BL21(DE3) cells. To screen for each mutation, four putative recombinant clones were randomly picked from the agar plate and overnight cultures were prepared for plasmid extraction. Subsequently, the putative plasmid that carried the desired mutant gene was identified through DNA sequencing. As the mutations were dispersed throughout sjIPNS gene, those mutations located near the 5’ end of the gene were sequenced using the forward primer OL48 which annealed to the T7 promoter. Accordingly, mutations found at the 3’ end of sjIPNS gene were sequenced using the reverse primer OL74 that annealed to the T7 terminator. This was illustrated in Fig. 4.19 which showed the details of the sequencing strategy and the comparisons of the respective sequenced regions of wildtype and mutant sjIPNS. Take for example, amino acid 19 of the wildtype sjIPNS sequence is coded by TCC for serine, and the mutant enzyme would have at the same position TTC codon that codes for phenylalanine. This site change is clearly shown in the electropherograms presented in Fig. 4.19a. The entire open reading frames of all the sjIPNS single mutants were also sequenced to verify that the mutants only contained the respective intended mutations and did not harbor other random mutations. Chapter Results Fig. 4.18 PCR mutagenesis of sjIPNS. (a) PCR mutagenic process; (b) Lane shows the λHindIII DNA markers. The reaction products derived from in vitro PCR site-directed mutagenesis of the 13 individual single mutations of sjIPNS are resolved by gel electrophoresis in lanes to 14. (a) Pfu polymerase sjIPNS Mutagenesis primer pair pET-SJ 6300bp (b) kbp 23.1 9.4 6.6 4.4 10 11 12 13 14 ~6.3kbp 2.3 2.0 129 Chapter Fig. 4.19 Results Sequence analysis of sjIPNS single mutants. Sequencing strategy used to verify the specific mutational changes introduced in sjIPNS is illustrated in the diagram below. Mutations analyzed via sequencing of the forward strand using OL48 that primed upstream of sjIPNS gene are shown in pink. Remaining mutations were verified using OL74 (shown in green) that primed downstream of sjIPNS gene and thus the reverse strand of the gene sequence was being analyzed. Sequencing results of all sjIPNS single mutants are presented in the table (a) and (b) below. The specific nucleotide regions sequenced are shown and the affected codons are underlined. Sequencing strategy: OL48 S19 A37 D112 E120 I156 Y160 sjIPNS gene A202 R242 H260 T308 T309 OL74 (a) Mutations analyzed by sequencing the forward strand of sjIPNS gene Mutation wild type sequence Mutant sequence Ser19Phe Ser (TCC) Phe (TTC) Ala (GCC) Cys (TGC) Ala37Cys Asp112Glu Asp (GAC) Glu (GAG) 130 Chapter Results Fig. 4.19 (ctd.) (a) Mutations analyzed by sequencing the forward strand of sjIPNS gene Mutation wild type sequence Mutant sequence Glu120Gly Glu (GAG) Gly (GGG) Ile (ATC) Leu (CTC) Tyr (TAC) Phe (TTC) Tyr (TAC) Leu (TTG) Ile156Leu Tyr160Phe Tyr160Leu 131 sjIPNS-Tyr160 (b)I Ile218 Mutant-Phe160 Asn250 Cys251 Val249 Met255 (b)II Ile218 Tyr160 Ile156 Ala37 (b)III Cys251 Asn250 Val249 Met255 Phe43 Phe160 Ala37 Ile156 Phe43 Phe 171 Mutant-Leu160 Phe 171 Ile33 Leu 164 Ala163 Ala30 Leu 164 Ala30 Leu164 Ala37 Phe160 Ile33 Ala163 Ala30 Phe263 (d)II Ile33 Ala163 Ala30 Phe263 Asn250 Asn250 Val249 Phe43 Phe171 Leu164 Ala37 Ile33 Leu160 Ala163 Ala30 Asn250 Val249 Phe43 Phe171 Leu164 Phe263 Asn250 Val249 Phe43 Tyr160 Phe263 (d)III Asn250 Ala37 Ala30 Phe43 Phe171 Val249 Phe171 Leu164 Ala163 Val249 Ala163 Ala30 Phe263 (d)I Leu 164 (c)III Phe43 Ile33 Ile33 Asn250 Val249 Tyr160 Val249 Phe43 Leu160 Ala37 Ile156 Phe 171 Phe263 (c)II Ala37 Asn250 Met255 Ala163 Asn250 Phe171 Leu164 Cys251 Ile33 Phe263 (c)I Ile218 Ala37 Phe160 Ile33 Ala163 Ala30 Phe43 Phe171 Leu164 Phe263 Fig. 4.36 (ctd.) Ala37 Ile33 Leu160 Ala163 Ala30 * For figure (c)I-III, all the residues analyzed are shown in space-filled representations except Phe263, Tyr160, substituted Leu160 and Phe160 which are shown in carbon atom representations. Chapter Results greater reduction in residue size was observed for leucine substitution (-26.7) than for phenylalanine substitution (-3.7) (Table 4.8) in which the aromatic ring is preserved with only the polar-OH being removed. From the space-filled analyses in Fig. 4.36c, d, it is apparent that Tyr160Leu substitution produces a greater cavity in the hydrophobic core of sjIPNS than Tyr160Phe substitution whereby the removal of the –OH group seems to be of negligible effect. Hydrophobic cores of most natural proteins are usually packed with rare occurrence of cavities and only small volume changes are tolerated. Hence, although leucine substitution at site 160 effectively removed the adverse interaction between the –OH group of Tyr160 and its hydrophobic surrounding, it also created another problem of forming sizable cavity in the hydrophobic core of sjIPNS. Thus, it seems that not only the hydrophobicity of the substituted side chain is important for site 160; the bulkiness of the side chain is also crucial to permit optimum hydrophobic contacts with the hydrophobic surrounding. Hence, multi-level constraints are observed at this buried site whereby the enhanced solubility of sjIPNS mutant Tyr160Phe entailed removal of the detrimental buried polar group effect and optimization of the local hydrophobic interactions ensuring maximal hydrophobic contacts to avoid cavity formation in the hydrophobic core. These allure to the difficulties in engineering changes in protein hydrophobic core. Superimposition analyses of the neighboring residues within 6Å from Tyr160 in sjIPNS (residues showed in blue) with the neighboring residues of corresponding Phe160 in slIPNS (residues shown in cyan) and Leu160 in scIPNS (residues showed in yellow) are shown in Fig. 4.36eI and II respectively. slIPNS was chosen as the representative for sfIPNS and nIPNS. Both slIPNS Phe160 and scIPNS Leu160 were observed to form the same hydrogen bonding interactions (highlighted in red dotted lines) as were observed for Tyr160 in sjIPNS (highlighted in cyan dotted lines). High conservedness, ranging from 70-90%, in the local milieu surrounding position 160 was observed between insoluble sjIPNS and the soluble IPNS isozymes (Fig. 4.30b). The only significant difference is the hydrophobicity of the side chains. Intuitively, it serves to highlight that the poor solubility of sjIPNS may be due to the poor choice of residue at this site where bulky hydrophobic residues are much preferred to facilitate Chapter Results hydrophobic interactions. However, a noteworthy point is that scIPNS is soluble although the presence of leucine at site 160 cause a cavity in this buried environment. Mutant Ala202Prohyperexposed Ala202 is the first residue of the solvent exposed loop13 which is a very short loop that forms a tight turn linking β6 and β7 (Fig. 4.37a). The methyl side chain of Ala202 is shown to be hyperexposed, extending fully into the solvent exterior with 59% solvent accessibility (Table 4.11). As such, only seven neighboring residues were elucidated within its 6Å vicinity and hydrophilic residues constituted 70% of these residues (Fig. 4.30b). The Ala202 does not form any hydrogen bonding interaction (Fig. 4.37bI). The conserved replacement of hydrophobic Ala202 with hydrophobic Pro202 besides causing a 24.1 and 3.4 value increase in side chain volume size and hydrophilicity respectively (Table 4.8), it did not result in any new hydrogen bond formation (Fig. 4.37bII). The increase in hydrophilicity at this hyperexposed site did not seem to improve the solubility of the sjIPNS mutant Ala202Pro. The substitution of the 5-membered ring structure of Pro202 may potentially introduce conformational strain to the sharp β-turn structure that it exists in. However, close examination revealed that two glycine residues are located at position 201 and 204 respectively. The small and simple structures of glycine residues may help to accommodate some of the rigidity caused by Pro202. Even so, the Ala202Pro substitution was not found to contribute favorably to improving the solubility of sjIPNS since mutant Ala202Pro remained insoluble at 25ºC. Superimposition analysis of the local environments of Ala202 in sjIPNS (neighboring residues in blue) and the corresponding Pro202 in soluble scIPNS (neighboring residues in yellow) showed high 90% conservedness in the identities and conformations of the neighboring residues (Fig. 4.37c). Pro202 in scIPNS is also not involved in any hydrogen bond interaction. The methyl side chain of sjIPNS Ala202 and the ring structure of scIPNS Pro202 Fig. 4.37 Structural analysis of Ala202Pro substitution in sjIPNS. (a) sjIPNS-Ala202 Mutant-Pro202 (b)I (b)II Loop12 β5 Thr200 Thr205 β7 Thr200 Thr205 Gly204 Gly204 β8 β6 Arg269 Loop13 Glu210 Arg269 Gly201 Asp203 Loop14 (b)IV (b)III Gln205 Thr205 Thr200 Arg269 Arg269 Thr200 Arg269 Asp203 Ala202 Gly204 Thr200 Gly204 Gly204 Pro202 Asp203 Pro202 (c) Gly201 Gly201 Ala202 Ala202 Glu210 Glu210 Glu210 Gly201 Glu210 Gly201 Asp203 Pro202 Ala202 Asp203 Chapter Results were shown to be similarly orientated. Apparently by changing the alanine residue in sjIPNS at position 202 to proline residue which is found in all soluble isozymes at the same position did not cause much perturbation to the local environment. Mutant Arg242Thr*exposed Arg242 is located very close to the turn region of loop17 that connects β10 and β11 (Fig. 4.38a). These two β-strands are part of the anti-parallel network of eight β-strands that forms the jelly-roll motif in IPNS. The long positively charged side chain of Arg242 points into the interior of the jelly-roll motif, exposing only its main chain atoms on the protein surface, thus justifying its partial solvent accessibility of 14% (Table 4.11). The side chain of Arg242 is embedded in a crowded environment consisting of 15 neighboring residues, 60% of which are hydrophilic residues (Fig. 4.30b). Detailed analysis revealed that the –CH2-CH2CH2-NH-C(NH2)NH2+ side chain of Arg242 is engaged in a bifurcate hydrogen bonding interaction with the main chain of Thr224 (β8) and form another additional hydrogen bond with the side chain of Asn228 (β9) (Fig. 4.38bI). Substitution of positively charged arginine with polar uncharged threonine at position 242 caused a drastic change in the hydrogen bonding network and side chain orientation. The three hydrogen bonds observed for wildtype Arg242 were eliminated and in place, a new hydrogen bond was predicted to form between the side chain of Thr242 and the side chain of a new bonding partner, Glu225 (highlighted in CPK color in Fig. 4.38bII). In addition, the new -CH(OH)CH3 side chain of Thr242 projects away from the interior of the jelly-roll motif into the solvent. Consequently, Thr242 becomes more exposed with 30% solvent accessibility. Conceivably, this reversal of side chain orientation may help to reduce possible steric strain on Arg242 in the wildtype, which is set in a highly crowded environment (Fig. 4.38bIII). As mentioned earlier, Arg242 is located in a relatively hydrophilic surrounding and examination of the neighboring residues revealed that another arginine is found at position 277 Fig. 4.38 Structural analysis of Arg242Thr substitution in sjIPNS. (a) sjIPNS-Arg242 Mutant-Thr242 (b)II (b)I Arg242 Loop17 β7 β9 β5 β10 Glu225 β12 Asn228 Arg242 β8 (c) Glu225 Arg277 β13 β11 Thr224 Thr224 Arg277 Asn228 Thr242 (b)III (b)IV Thr224 Thr224 Thr224 Arg277 Glu225 Glu225 Glu225 Arg242 Thr242 Asn228 Arg242 Arg277 Asn228 Thr242 Arg277 Asn228 Chapter Results (side chain highlighted in CPK color in Fig. 4.38b) which happens to be involved in substrate binding in the catalysis of IPNS. In fact, the two positively charged (NH2)NH2+ groups of Arg242 and Arg277 were determined to be about 5.05Å apart using the manipulation tools of SPdbV program. Such close proximity between two positively charged groups is likely to be unfavorable due to same charge repulsion. Removal of the positively charged arginine at position 242 would plausibly eliminate this charge repulsion problem. The other neighboring charged residue is Glu225 that is not involved in any bonding interactions in wildtype sjIPNS and is one of the residues determined to be lacking proper hydrogen bonding by the SPdbV program. However, this bonding problem was alleviated in mutant Arg242Thr whereby the side chain of substituted threonine formed a new hydrogen bond with the side chain of negatively charged Glu225. Hence, an interesting scenario occurred for Arg242Thr substitution in sjIPNS. Although the replacement incurred a major change in hydrogen bonding network, this seems to be compensated by the resultant optimization of the region’s charge-charge and hydrogen bonding interactions since the substitution led to improved sjIPNS solubility. In soluble scIPNS, threonine exists at position 242 (highlighted in green in Fig. 4.38c) instead of arginine as seen in sjIPNS (highlighted in pink in Fig. 4.38c). The side chain of Thr242 in scIPNS projects into the solvent while the side chain of Arg242 sjIPNS is buried in the jelly-roll motif. Due to the different orientations of side chains, Thr242 in scIPNS is in a much less dense environment consisting of only ten neighboring residues compared to the fifteen residues surrounding Arg242 in sjIPNS (Fig. 4.30b). Part of the local milieu of sjIPNS Arg242 includes residues from the jelly-roll motif which are however, not in close proximity to Thr242 in scIPNS (neighboring residues in sjIPNS and scIPNS are shown in blue and yellow respectively in Fig. 4.38c). One of these residues is Arg277 which is found to offer unfavorable charge interaction with the side chain of Arg242 in sjIPNS. Thr242 in scIPNS does not form any hydrogen bond with its neighboring residues, unlike Arg242 in sjIPNS which is engaged in three hydrogen bonding network. Although the side chains of threonine residues in sjIPNS mutant Arg242Thr and scPNS shared the same Chapter Results conformation, only the former is predicted to form one hydrogen bond with Glu225. This is likely due to the different side chain orientation of Glu225 in sjIPNS and scIPNS (Fig. 4.38c). Extrapolating from the analysis of Thr242 in soluble scIPNS isozyme, it seems that the loss of the three hydrogen bonds accompanying the Arg242Thr substitution in sjIPNS mutant Arg242Thr is unlikely to be structurally detrimental. Mutant His260Asnexposed His260 is located in the solvent exposed loop19 connecting α8 and β12 (Fig. 4.39a). The positively charged side chain of His260 is 33% solvent accessible as it extends into the solvent exterior (Table 4.11). Detailed analysis of the residues surrounding His260 within 6Å showed that the ratio of hydrophilic to hydrophobic residues is 40:60 (Fig. 4.30b). Close scrutiny revealed the presence of two other positively charged residues (His257 and Lys305) and a negatively charged residue (Asp261) near His260 (all highlighted in CPK color in Fig. 4.39b). Hydrogen bond analysis showed that the main chain of His260 forms a single hydrogen bond with the main chain of Gly256 (Fig. 4.39bI). Substitution of the positively charged side chain of His260 with the polar uncharged side chain of Asn260 resulted in a new hydrogen bond being formed between the -NH2 group of Asn260 and the C=O group of Asn306 (Fig.4.39bII). Interestingly, the introduction of a new hydrogen bond has no effect on improving the solubility of sjIPNS. In fact, analysis of the equivalent Asn260 in scIPNS (shown in green in Fig. 4.39c) showed that the residue forms the same hydrogen bond network (highlighted by red dotted lines) as what has been predicted for Asn260 in sjIPNS mutant His260Asn. Apparently, the charged His257 and Asp261 residues are also present in the local environment of scIPNS whereas positively charged arginine instead of positively charged lysine is found at site 305 (neighboring residues of scIPNS and sjIPNS are shown in yellow and blue respectively in Fig.4.39c). Hence, the residues at positions 260 in both sjIPNS and scIPNS are located in densely charged milieu. Perhaps, the Fig. 4.39 Structural analysis of His260Asn substitution in sjIPNS. (a) Loop20 Mutant-Asn260 sjIPNS-His260 (b)I (b)II β13 β12 Gly256 Gly256 Asp261 His257 α9 Asp261 His257 Loop19 α8 Asn260 Asn306 Asn306 His260 His260 Loop21 Lys305 Lys305 (c) (b)IV (b)III Gly256 Ala256 His257 Asp 261 Asn 260 Asn306 His260 Gly256 His257 Gly256 Asp261 Asn306 His257 Asn306 Asp261 Asn260 His260 Glu303 Lys305 Lys305 Arg305 Lys305 Chapter Results His260Asn substitution in sjIPNS mutant His260Asn which only incurs negligible hydrophobicity index change (-0.3) did not significantly alter the local charge environment. Mutant Thr308Ala*exposed Thr308 is located in loop21, which happens to be the longest loop in sjIPNS composing of 26 amino acid residues and also the least conserved among the bacterial IPNS isozymes analyzed (Table 4.10). Only nine residues were elucidated within 6Å from Thr308. Survey of the neighboring residues shows 60% of the residues are hydrophilic in nature (Fig. 4.30b). The remaining neighboring residues that are hydrophobic include Val291, Ile292, Pro294 and Pro307 (Fig. 4.40bIII). Interestingly, Val291, Ile292 and Pro294 are located in adjoining positions in region of loop21 that is slightly shielded from the exterior and form a short contiguous hydrophobic patch. Although the 30% solvent exposed -CH-CH3(OH) polar side chain of Thr308 occurs on the surface of sjIPNS (Table 4.11), its side chain is oriented towards the hydrophobic patch, especially its -OH group is found directed towards the hydrophobic five-membered ring of Pro294. In fact, the -OH group of Thr308 is only 4.45Å from the ring structure of Pro294. Detailed analysis showed that Thr308 does not participate in any hydrogen bond formation (Fig. 4.40bI). Replacing the -CH-CH3(OH) polar side chain of threonine with the hydrophobic methyl group of alanine at position 308 did not form any new hydrogen bond nor cause much perturbation to the region (Fig. 4.40bII, IV). However, Thr308 is one of the residues shown to be lacking in proper hydrogen bonding interaction by the SPdbV program. It is thus postulated that alanine substitution would help to remove the unfavorable interaction between the free polar -OH group of wildtype Thr308 and the elucidated hydrophobic patch, especially with the hydrophobic side chain of Pro294. The substituted hydrophobic Ala308 in mutant Thr308Ala is potentially able to make more favorable contacts with the hydrophobic patch, thereby optimizing the hydrophobic interactions in the region. Hence, the presence of a local hydrophobic patch at site 308 may account for why the introduction of a more hydrophobic Fig. 4. 40 Structural analysis of Thr308Ala substitution in sjIPNS mutant 308.* Mutant-Ala308 sjIPNS-Thr308 (a) (b)I β13 (b)II α9 Val291 Ile292 Ile292 Pro307 Thr308 Pro307 Ala308 Thr308 Pro294 Pro294 Glu293 Loop21 (c) Thr309 Val291 Thr309 Glu293 (b)IV (b)III Ser310 Val291 Val291 Leu309 Thr309 Ile292 Thr308 Ile292 His257 Thr308 Glu307 Pro307 Thr309 Val291 Ile292 Ala308 Thr309 Ala308 Pro307 Pro294 Glu293 Pro294 Asn306 * For figure (b)III and IV, Glu293 is not shown in the space-filled representations to allow better visualization of the residues beneath. Pro307 Pro294 Chapter Results residue, incurring a significant 2.5 value increase in the side chain hydrophobicity index (Table 4.8), at this solvent exposed position in sjIPNS is accommodated. Although loop21 is the least conserved loop among the bacterial IPNS isozymes, the neighboring residues surrounding site 308 in sjIPNS and scIPNS shows 80% conservedness in identities (Fig. 4.30b) and high homology in amino acid side chain conformations (Fig. 4.40c, sjIPNS residues in blue and scIPNS residues in yellow). Val291, Ile292 and Pro294 which form the continuous hydrophobic patch enclosing site 308 in sjIPNS are also conserved in scIPNS (Fig. 4.40c). Both Ala308 in scIPNS (showed in green) and Thr308 in sjIPNS (showed in pink) not engage in any hydrogen bonding interaction. The side chain of both scIPNS Ala308 and sjIPNS Thr308 project towards the hydrophobic patch, the only difference being the hydrophobicity of the side chains. Thus, the unfavorable interaction between the polar side chain of Thr308 and the hydrophobic patch in sjIPNS may cause it to be different in solubility from scIPNS which has hydrophobic alanine residue at the same position. This phenomenon is also observed for the Ser19Phe substitution at site 19 in sjIPNS. Mutant Thr309Val/ Mutant Thr309Leu*exposed Thr309 is the residue next to Thr308, hence Thr309 is also located near the C-terminal end of loop21, the longest loop in sjIPNS (Fig. 4.41a). 70% of the 12 residues elucidated within 6Å from Thr309 consist of hydrophilic residues. Therefore like Thr308, Thr309 is situated in a more hydrophilic environment (Fig. 4.30b). However, unlike Thr308 which exhibits 30% solvent accessibility, Thr309 is more buried with only 16% of its surface area exposed to the solvent (Table 4.11). The -CH-CH3(OH) polar side chain of Thr309 is found projecting towards the interior of sjIPNS (Fig. 4.41bI). Detailed analysis of the site revealed an interesting observation that as many as three tyrosine residues, Tyr256, Tyr311 and Tyr314, are located near Thr309 (side chains showed in CPK color in Fig. 4.41b-d). The side chains of these three tyrosine residues are held in such a way that the carbon atoms of the three aromatic rings come together to form a contiguous hydrophobic patch, which is distinctively shown in Fig. 4.41 Structural analyses of Thr309Val and Thr309Leu substitutions in sjIPNS. (e)I (a) (e)II Glu313 Asp313 β14 Tyr314 Tyr311 Tyr256 α9 Ser310 Thr312 Tyr311 Leu309 Thr309 Tyr256 Val311 Thr309 His257 Tyr259 Leu260 Tyr314 Asn 308 Thr309 Thr308 Ala310 Pro307 Glu307 Ile292 Met294 Glu293 Loop21 Superimposed sjIPNS & slIPNS Thr308 Ala308 Superimposed sjIPNS & scIPNS sjIPNS-Thr309 Mutant-Val309 (b)I Mutant-Leu309 (b)III (b)II Tyr314 Tyr256 Tyr314 Tyr314 Tyr311 Thr309 Tyr311 Tyr311 Tyr256 Leu309 Val309 Tyr256 (c)I Thr309 Tyr311 Tyr314 (c)III (c)II Leu309 Val309 Tyr314 Tyr311 Tyr311 Tyr314 Tyr256 Tyr256 Tyr256 (d)I (d)III (d)II Tyr311 Tyr311 Tyr314 Tyr314 Thr309 Leu309 Tyr314 Val309 Tyr256 Tyr256 Fig. 4.41 (ctd.) Chapter Results Fig. 4.41cI. As in the case of Thr308, the polar uncharged side chain of Thr309 is directed towards the hydrophobic patch. To more clearly depict the side chain interaction of Thr309 with the hydrophobic patch formed by the aromatic rings of the three tyrosines, two different views of the site in space-filled representations are shown in Fig. 4.41cI and dI. Hydrogen bond analysis indicated that Thr309 is not involved in any hydrogen bonding interaction. It is peculiar that bulky hydrophobic residues are found at position 309 in all the soluble IPNS isozymes, i.e, leucine in scIPNS, valine in slIPNS and nIPNS, and isoleucine in sfIPNS. Contrastingly, polar uncharged threonine residue is found at the same position in sjIPNS. In this study, Thr309 in sjIPNS has been replaced by hydrophobic valine and leucine residue in sjIPNS mutant Thr309Val and Thr309Leu respectively (Fig. 4.41bII, III). Both changes did not bring about any new hydrogen bond formation nor cause much perturbation to the local environment. The noticeable changes accompanying both substitutions are the drastic increase in the hydrophobicity indices and the bulkiness of the side chains (Table 4.8). Substituted Leu309 in mutant Thr309Leu is observed to incur a greater increase in residue volume size (+50.6) than substituted Val309 in mutant Thr309Val (+23.9). The hydrophobic side chains of both Val309 and Leu309 are also found to project towards the hydrophobic patch formed by the aromatic rings of the three tyrosine residues defined earlier on. Similar to what was observed for mutant Thr308Ala, the removal of the -CH-CH3(OH) polar side chain of Thr309 would likely eliminate the unfavorable interaction between the hydrophilic -OH group and the hydrophobic patch (Fig. 4.41bI, dI). The hydrophobic side chains of substituted Val309 and Leu309 would in turn interact favorably with the hydrophobic patch (Fig. 4.41cIIIII, dII-III). In particular, the bulkier side chain of Leu309 actually fills up the hydrophobic cavity (Fig. 4.41cIII, dIII). Therefore, it seems that optimization of the local hydrophobic interactions at site 309 maybe a crucial determinant for the improved solubility observed for mutant Thr309Val and Thr309Leu. Once again, the placement of a hydrophobic residue at the solvent exposed surface of sjIPNS is accepted due to the existence of a local hydrophobic cavity. Chapter Results The Thr309Val and Thr309Leu substitutions in sjIPNS were carried out based on the identities of the corresponding amino acid residues found naturally occurring in soluble scIPNS, slIPNS and nIPNS respectively. As such, the neighboring residues within 6Å of site 309 in sjIPNS (residues showed in blue in Fig. 4.41eI, II) were compared with the corresponding neighboring residues of the same sites in sIIPNS (residues showed in cyan in Fig. 4.41eI) and scIPNS (residues showed in yellow in Fig. 4.41eII). slIPNS is chosen as the representative for nIPNS. Generally, the local environment of sjIPNS Thr309 shows 70% homology with that of slIPNS and scIPNS (Fig. 4.30b). Amongst the neighboring residues found conserved between the three lPNS isozymes are the three tyrosine residues, Tyr256, Tyr311 and Tyr314 (according to sjIPNS numbering and labeled in white in Fig. 4.41eI, II). Hence, the hydrophobic patch observed in sjIPNS is also present in slIPNS and scIPNS. The corresponding residues of sjIPNS Thr309 in sIIPNS (Val311, according to sIIPNS numbering and shown in green in Fig. 4.41eI) and scIPNS (Leu309, according to scIPNS numbering and shown in green in Fig. 4. 41eII) also have their side chains projecting toward the hydrophobic patch. The only difference being the hydrophobicity of the side chain of sjIPNS Thr309 and that of Val311 and leu309 in sIIPNS and scIPNS respectively. Thus, the same phenomenon observed for the substitutions at prior analyzed solvent exposed sites 19 and 308 was also seen at site 309 whereby the unfavorable interaction between the polar side chain of Thr309 and its local hydrophobic patch may cause sjIPNS to be less soluble compared to the soluble isozymes which all have hydrophobic residues at the same position. [...]... and were all found mainly in the insoluble protein fractions (results not shown) Hence, no differences were observed in the expression profiles of wild type and mutant sjIPNS proteins at 37 °C and 30 °C whereby all were insoluble Interesting variations in the production of soluble wildtype and mutant sjIPNS proteins were observed at 28°C (Fig 4.20a) For instance, while only 1-4% of wildtype sjIPNS and. .. Summary of scIPNS mutants constructed Amino acid position in scIPNS Amino acid residue in scIPNS Amino acid residue in sjIPNS scIPNS single mutant constructed 120 Gly Glu Gly120Glu 30 9 Leu Thr Leu309Thr Fig 4 23 Gel electrophoresis of the in vitro PCR site-directed mutagenesis reactions for scIPNS single mutants and sequence confirmation of the respective mutants (a) Lane 1 shows the λHindIII DNA marker... mutations is additive and what consequences ensue when too many mutations were introduced Thus, to understand the effects of combined mutations on the expression of soluble sjIPNS, more site-directed mutagenesis were performed to construct sjIPNS double and triple mutants for expression analysis in E coli BL21(DE3) 4 .3. 4.1 Mutagenesis, selection and sequence confirmation of sjIPNS double and triple mutants... form during protein folding Using the Chou and Fasman index (Chou and Fasman, 1978), five amino acid residues, aspartate, asparagine, proline, glycine and serine, have been proposed to be involved in forming the turns of proteins Thus, if a protein contains a high proportion of these residues, the protein may be inclined to form inclusion bodies due to slower folding rate (Wilkinson and Harrison, 1991)... Thr309Val) and leucine (mutant Thr309Leu) substitutions at amino acid position 30 9 with + 23. 9 and +50.6 volume change respectively resulted in equally soluble mutant proteins Hence, the magnitude of residue volume change seems to be important for the outcomes of substitutions at amino acid position 160 but not for position 30 9 Analysis of the changes in the hydrophobic and hydrophilic properties of the amino. .. of the determinant sites that were not tested here 4.4 Characterization of sjIPNS mutants via computational analysis The varied effects of different amino acid substitutions in sjIPNS on its solubility unveiled the intricate relationship between protein sequence and structure, in that only specific changes in the primary sequence could affect protein folding and unfolding, resulting in either more... sequencing using primer OL48 which recognize the T7 promoter of the vector sequence On the other hand, sequencing using reverse primer OL74 annealing to the T7-terminator of the vector was used to confirm Thr308Ala and/ or Thr309Val substitutions at the 3 end of sjIPNS gene Electropherograms from the sequencing of the various sjIPNS multiple mutants are shown in Fig 4.21b 4 .3. 4.2 Expression analysis of. .. sjIPNS double and triple mutants The expression of sjIPNS double and triple mutants in E coli BL21(DE3) was carried out at 37 °C to 18°C as described previously and compared with the expression of wildtype sjIPNS No notable differences between the expression of the two double mutants and the triple mutant, and wildtype sjIPNS were seen at 37 °C and 30 °C whereby all were produced in the insoluble protein... purely in terms of certain amino acid properties However, the local environments of the substituted residues also play an important role in modulating the outcomes of the amino acid replacements For example, an increase in residue volume size in a crowded local environment may result in steric strain As such, it is important to include the structural analysis of the interrelated local environments of the... mutagenesis to construct the sjIPNS Chapter 4 Results Table 4.5 Summary of sjIPNS double and triple mutants constructed Number of amino acid substitutions Specific mutations DNA template used for PCR mutagenesis Recombinant pET24a-Thr309Val sjIPNS mutant gene Glu120Gly Thr309Val Double mutant Name of resultant mutant Glu120Gly/Thr309Val Thr308Ala Thr309Val Recombinant pET24a-Thr309Val sjIPNS mutant gene . changes are Chapter 4 Results 125 Fig. 4.17 Amino acid sequence alignment of insoluble sjIPNS and soluble bacterial IPNS isozymes (scIPNS, sfIPNS, slIPNS and nIPNS). Numbering of amino. our investigation. Position in sjIPNS sjIPNS Soluble bacterial IPNS isozymes (scIPNS, sfIPNS, slIPNS & nIPNS) Amino acid residue Amino acid property Amino acid residue Amino acid. upstream of sjIPNS gene are shown in pink. Remaining mutations were verified using OL74 (shown in green) that primed downstream of sjIPNS gene and thus the reverse strand of the gene sequence was