Characterization of the molten globule state of retinol-binding protein using a molecular dynamics simulation approach Emanuele Paci 1 , Lesley H. Greene 2 , Rachel M. Jones 2 and Lorna J. Smith 2 1 Institute of Molecular Biophysics, School of Physics and Astronomy, University of Leeds, UK 2 Department of Chemistry and Oxford Centre for Molecular Sciences, Chemistry Research Laboratory, University of Oxford, UK The detailed characterization of molten globule states of proteins continues to be an area of intense research activity and interest (for reviews see [1,2]). This is in part because these equilibrium partially folded states have been found to have many similarities to kinetic protein folding intermediates. As such, the properties of these states can therefore give important insights into the determinants of protein structure and folding [2]. Molten globule states of proteins are also postula- ted to be involved in a range of important physiologi- cal processes, including the insertion of proteins into membranes, the release of bound ligands and aggrega- tion [1]. In this latter area, molten globule-like species are thought in some systems to be precursors of amy- loid fibril formation [3,4]. Molten globule ensembles are characterized by hav- ing a pronounced amount of secondary structure, in a compact state that lacks most of the specific tertiary interactions coming from tightly packed side chain groups [1,2]. One of the molten globule states that has been studied in the most depth is that of a-lactalbumin [5]. In this case, it has been possible using nuclear magnetic resonance (NMR) methods to gain a residue specific picture of the noncooperative unfolding of the molten globule during denaturation with urea [6–8]; data from these experiments have also been used as Keywords lipocalin; molecular dynamics; molten globule; protein folding; retinol-binding protein Correspondence L. J. Smith, Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK Fax: +44 1865 285002 Tel: +44 1865 275961 E-mail: lorna.smith@chem.ox.ac.uk E. Paci, Institute of Molecular Biophysics, School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, UK Tel: +44 113 3433806 E-mail: e.paci@leeds.ac.uk (Received 25 May 2005, revised 12 July 2005, accepted 3 August 2005) doi:10.1111/j.1742-4658.2005.04898.x Retinol-binding protein transports retinol, and circulates in the plasma as a macromolecular complex with the protein transthyretin. Under acidic con- ditions retinol-binding protein undergoes a transition to the molten globule state, and releases the bound retinol ligand. A biased molecular dynamics simulation method has been used to generate models for the ensemble of conformers populated within this molten globule state. Simulation con- formers, with a radius of gyration at least 1.1 A ˚ greater than that of the native state, contain on average 37% b-sheet secondary structure. In these conformers the central regions of the two orthogonal b-sheets that make up the b-barrel in the native protein are highly persistent. However, there are sizable fluctuations for residues in the outer regions of the b-sheets, and large variations in side chain packing even in the protein core. Signifi- cant conformational changes are seen in the simulation conformers for resi- dues 85–104 (b-strands E and F and the E-F loop). These changes give an opening of the retinol-binding site. Comparisons with experimental data suggest that the unfolding in this region may provide a mechanism by which the complex of retinol-binding protein and transthyretin dissociates, and retinol is released at the cell surface. Abbreviations ANS, 8-anilino-1-napthalenesulphonate; MD, molecular dynamics; RBP, retinol-binding protein; R g , radius of gyration; RMSD, root-mean- square deviation; TTR, transthyretin. 4826 FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS restraints in a novel approach to determine the free energy landscape of this molten globule [9]. In the majority of the proteins whose molten globule states have been characterized, both by experimental and the- oretical methods, predominantly a-helical secondary structure persists in the molten globule state [10–13]. In contrast, in this paper we concentrate on a protein which is rich in b-sheet secondary structure in the native state, and which retains the majority of this b-sheet secondary structure in the molten globule state, human serum retinol-binding protein (RBP). RBP is a member of the lipocalin superfamily [14]. The proteins in this superfamily adopt a similar fold; an eight-stranded up-and-down b-barrel with a C-ter- minal helix. However, they have a wide range of func- tions, and high levels of sequence divergence, with many members sharing under 20% sequence identity [14,15]. The lipocalins are widely distributed through- out the eukaryotic and prokaryotic kingdoms [16,17]. Many of the lipocalins act as transporters for small nonpolar ligands, such as retinoids, haem, phero- mones, lipids, prostaglandins and pigments [18]. RBP transports all-trans-retinol (vitamin A) from its storage sites in the liver to target tissues [19]. It is postulated that the local decrease in pH at the surface membrane of target cells triggers the release of retinol, by a mech- anism that is dependent on the conversion of RBP to a molten globule state [20,21]. It is possible that similar mechanisms may prompt ligand release for other mem- bers of the lipocalin superfamily. For example, the release of lipid ligands by human tear lipocalins under acidic conditions is thought to be associated with a transition to the molten globule state [22,23]. The molten globule state of RBP, formed under acidic conditions, has been shown to exhibit the key character- istics typical of these partially folded states. Stoke’s radii, from diffusion coefficient measurements, have demonstrated that the molten globule retains a compact fold [20]. The mean molecular dimensions of the parti- ally folded ensemble are only 13% larger than those of the native state. Far- and near-UV circular dichroism (CD) spectra show that the protein contains a significant level of secondary structure, but has a considerable level of disorder in side chain packing, respectively [20,24,25]. Obtaining detailed information at an atomic level about the molten globule state using techniques such as NMR spectroscopy is challenging. Partially folded states such as molten globules are ensemble of interconverting con- formers [26]. Slow interconversion between populated conformers gives rise to broadened NMR resonances, while averaging of chemical shifts across the populated ensemble gives a limited chemical shift dispersion [6,27]. Therefore, to develop a detailed model to further our understanding of the molten globule state of RBP, in vitro and in vivo experimental studies have been com- plemented with a molecular dynamics (MD) simulation study. The results of this are reported here. It is not possible to explore adequately the conform- ational space accessible to partially folded proteins, within the simulation timescale currently accessible to conventional MD simulations of proteins in explicit solvent. The exploration of non-native conformations is therefore usually achieved, either by using very high temperatures in the simulations, or by introducing a suitable perturbation in a biased MD simulation, often using implicit solvent models (for a review of this topic see, e.g [28,29]). Explicitly modelling the perturbation induced by a change in the solution pH would not prompt the transition from the native to the molten globule state, on a timescale which can be directly simulated. In this work therefore we have used three different perturbations in turn. One perturbation forces an increase in the protein radius of gyration, the sec- ond perturbation induces the breaking of native con- tacts in the structure and the third perturbation is aimed to speed up the exploration of diverse (in terms of mutual RMSD) conformations. These various per- turbations are applied using a particularly ‘soft’ time- dependent bias [30,31], designed to generate low energy pathways in the conformational space. The large num- ber of diverse and moderately non-native conforma- tions generated with this biased molecular dynamics approach are then used as initial conformations for unperturbed, room temperature simulations. This method allowed us to explore local free energy minima in a broad region of the conformational space close to the native state. The approach is designed to provide a qualitative map of the free energy landscape, in a region of the conformation space compatible with the experimental knowledge of the molten globule state. By applying this sampling approach to RBP, we are able to identify a broad basin of low energy partially folded conformers that are compatible with the avail- able experimental data [20,24,25,32–34]. These con- formers provide a model for the molten globule state of RBP that allows us to gain insight into the determi- nants of protein folding and the mechanism of retinol delivery and release, an important physiological prob- lem which remains unresolved. Results and Discussion Sampling of conformational space As described in the Experimental procedures section, a biased MD simulation method has been used to E. Paci et al. MD simulations of the RBP molten globule state FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS 4827 generate 160 diverse configurations of RBP. Each of these was then used as an initial configuration in an unbiased MD simulation of 1.2-ns length. Figure 1(A) shows a plot of the heavy atom RMSD (root-mean- square deviation) from the X-ray structure as a func- tion of the radius of gyration (R g ), for structures taken every 10 ps through the 160 unbiased MD simulations. These data demonstrate the broadness of the conform- ational space explored in the study. Analysis of the range of RMSD values seen for the MD conformers shows that there are three distinct peaks in the RMSD distribution (Fig. 1B). These correspond to different levels of unfolding. To aid the analysis, the conformers have been divided into three groups on the basis of their RMSD values. Conformers in group 1 have an RMSD less than 4 A ˚ and an R g less than 16.5 A ˚ . Group 2 conformers have an RMSD in the range 4–7 A ˚ and an R g in the range 16.5–17.7 A ˚ , while group 3 conformers have an RMSD above 7 A ˚ and an R g greater than 17 A ˚ . The characteristics of the group 1 conformers are very native-like, in keeping with their low RMSD values (< 4 A ˚ ). For example, 81 of the residues that are in regions of secondary structure in the native protein have secondary structure popula- tions greater than 0.8 in the group 1 ensemble of con- formers. We therefore focus our attention on the conformers in groups 2 and 3 that display a greater level of unfolding. The native state structure of RBP has an R g of 15.9 A ˚ . The 13% increase in molecular dimensions seen experimentally on forming the molten globule state would correspond to an effective R g of 18 A ˚ for the molten globule ensemble. The group 3 conformers, with an R g in the range 17–20 A ˚ , show an appropriate level of expansion on average for the mol- ten globule state (Fig. 1A). The properties of this group of conformers have therefore particularly been compared with experimental data for this state. Secondary structure persistence The b-barrel in the native structure of RBP consists of eight b-strands (A-H) [35]. These are arranged in two orthogonal b-sheets, with some of the b-strands being involved in both of the sheets. The first sheet consists of strands ABCDEF, and the second sheet contains strands EFGHA (Fig. 2). The native structure also contains an a-helix (residues 146–158), which packs onto the b-sheet formed by the second set of strands. All of the b-strands present in native RBP show a high level of persistence in the group 2 conformers (Figs 2 and 3). However, in many of the structures some of these strands are reduced in length, or have irregularit- ies compared to those in the native state. For example, for strand F (residues 100–109) the mean b-sheet populations for the first five residues are only 0.05– 0.15. For strand E (residues 85–92) the mean b-sheet populations for the terminal residues 85 and 91–92 are 0.05–0.13, while those for residues 86–90 are 0.71–0.84. The disruption of this b-strand results predominantly 0 5 10 15 20 Å 0 0.1 0.2 0.3 0.4 0.5 normalized probability RMSD R g B 15 16 17 18 19 20 R g (Å) 0 2 4 6 8 10 12 RMSD (Å) Group2 Group3 Group1 A Fig. 1. (A) Relationship between the protein radius of gyration and the RMSD value from the X-ray structure of native RBP, for the 16 000 conformers taken at 10-ps intervals along the unbiased simulations. The radius of gyration and RMSD values are calculated using all heavy atoms. The black diamond corresponds to the native state following the 2 ns equilibration simulation. The definitions of the three groups of conformers used in the analysis are shown. (B) The distribution of radius of gyration (filled bars) and RMSD values (open bars) across the 16 000 simulation conformers. MD simulations of the RBP molten globule state E. Paci et al. 4828 FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS from the loss of hydrogen bonds between strands E and F, although in some structures the hydrogen bonds between strand D and E are also missing. The a-helix also shows a high level of persistence in the group 2 conformers, with a-helical populations in the range 0.59–0.99 for the residues involved. In the group 3 conformers the changes are more pronounced (Figs 2 and 3, and Fig. 1 in the supple- mentary material). All the b-strands now show reduc- tions in length compared to those in the native structure. Even strand H (residues 129–138), one of the particularly persistent strands, is reduced in length by three residues in more than half of the structures, with residues 129 and 130 having b-sheet populations of less than 0.1. In addition, in group 3 strand E and the first half of strand F are almost completely lost. Residues 85–92 and 100–104 show b-sheet populations of less than 0.3. A significant disorder is seen across the ensemble of group 3 conformers in the region con- taining strand E, the first part of strand F and the con- necting E–F loop (residues 85–104). The changes in this region correlate with results from crystallographic studies of bovine RBP. These report a conformational change in the E–F loop at low pH [34]. In addition, changes in this region were reported in a simulation of the apo form of RBP reported previously [36]. In almost all the group 3 conformers, however, a central region of the b-sheets is preserved. Residues in the central regions of strands B, C, D, G and H and part of strand A have b-strand populations greater than 0.9. A persistent section comprising the central regions of strands B, C and D together with part of strand A in b-sheet 1, and the C-terminal region of strand F with the central parts of strands G and H in b-sheet 2, have residues with b-strand populations greater than 0.8 (Figs 2 and 3). Hence in the group 3 conformers the central area of each of the two ortho- gonal b-sheets, that make up the b-barrel in the native protein, are retained. This is interesting as the two parts of the polypeptide chain that form these persist- ent central regions of the b-sheets have closely similar amino acid sequences. In particular, the sequence of RBP contains an internal repeat with residues 36–83 (includes b-strands BCD) and 96–141 (includes b-strands FGH) having 34% identity [35]. This may account, at least in part, for the similar behaviour of these regions in the simulations. The central region of the a-helix is also very persistent in the group 3 A B C D E F G H A B C Fig. 2. (A and B) The X-ray structure of human serum retinol-binding protein [35]. (A) The b-strands are labelled, those in b-sheet 1 are shown in red and those in b-sheet 2 are shown in cyan. The a-helix is blue and the retinol is magenta. (B) Only the residues that have a sec- ondary structure persistence greater than 0.8 in the group 3 conformers (Fig. 3) are coloured. The figure was generated using the program MOLSCRIPT [53]. (C) Backbone trace of representative structures from the three groups of simulation conformers (left, group 1; centre, group 2; right, group 3). In each case the average structure over the cluster centres is shown in red. E. Paci et al. MD simulations of the RBP molten globule state FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS 4829 conformers, residues 151–156 having a-helical popula- tions greater than 0.9. Experimental estimates of secondary structure con- tent from far-UV CD spectra give 45 and 40% b-sheet for the native and molten globule states of RBP, respectively [26]. The b-sheet estimate for the native- state is in close accord with that observed in the X-ray structure (46%) [35]. In the group 3 simulation con- formers, 53 residues have a b-sheet population of 0.60 or greater, and 11 residues have a b-sheet population in the range 0.40–0.60. Taken together this corres- ponds to 37% of residues in b-strand secondary struc- ture, a value similar to that seen experimentally for the molten globule state. The experimental CD data show that there is an increase in a-helical secondary struc- ture on forming the molten globule state (8% native; 24% molten globule [25]). A large increase in a-helical secondary structure is not observed, on average, in the simulation conformers. This difference may reflect sampling and force field limitations in the simulations, and the difficulty of interpreting experimental CD data in a quantitative fashion. The difference may also reflect the fact that we do not model explicitly the con- ditions under which the molten globule is stable in the simulations, but rather identify conformers that are low in energy under native conditions. However, although there is not a large increase in a-helical sec- ondary structure, the native state a-helix for residues 146–158 is essentially retained in all the simulation conformers. In addition, in some of the conformers, particularly those in group 2, turns, some of a helical character, do form for residues 93–96. These residues are in the loop connecting strands E and F in the native protein, a region where the native structure is significantly disrupted in the simulations. It is therefore possible that this is the part of the RBP sequence that forms non-native helical secondary structure when the molten globule state is adopted. Variations in side chain packing Despite the high persistence of the central regions of b-sheet secondary structure even in the conformers in group 3, significant changes are observed in the 0 0.2 0.4 0.6 0.8 probability 02040 60 80 100 120 140 160 residue number 0 0.2 0.4 0.6 0.8 A BC D E FGH Fig. 3. Fraction of the simulation conformers belonging to groups 2 (upper panel) and 3 (lower panel) in which certain secondary structure elements are present. Secondary structure was calculated using the program DSSPcont [54] which identifies regions of secondary structure through an analysis of hydrogen bonding patterns. b-sheet secondary structure is shown with open bars, helical (a,3 10 and p) secondary structure is shown with filled black bars, and turns and bends are shown with grey bars. The secondary structure present in the native state of RBP is indicated at the top of the figure, with the b-strands labelled A–H (open bars, b-strands; filled bars, a-helices). MD simulations of the RBP molten globule state E. Paci et al. 4830 FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS packing of hydrophobic side chains. The distances between pairs of aromatic side chains and between aromatic and other hydrophobic side chains, that are close in the native state, have been analyzed in the simulation conformers. Some representative examples of the distance distributions for conformers in groups 2 and 3 are shown in Fig. 4. A very broad distribution of distances across the simulation conformers is seen when one of the side chains involved is from residues 85–104. This is the region that forms strand E and part of strand F in the native structure, and is disor- dered in many of the simulation conformers. For example, the distance Tyr90–Met73 ranges from 3.3 to 46.6 A ˚ , while the distance Phe36–Tyr90 varies from 5.9 to 52.0 A ˚ in the group 3 conformers (Fig. 4). Fluctuations in the distances are seen even for resi- dues in regions where the native structure is retained to a significant extent. Here though, the variations are more limited. Thus for Phe20–Phe137 and for Phe36– Tyr133 the distance ranges are 3.2–15.3 A ˚ and 2.9–13.8 A ˚ , respectively, in the group 3 conformers (Fig. 4). These changes in the packing of aromatic side chains in the group 3 conformers are in accord with the experimental loss of a near-UV CD spectrum on forming the molten globule state of RBP [20,24,25]. These experimental data reflect the absence of fixed asymmetric environments for aromatic and cysteine residues in the molten globule. It is interesting that relatively short distances are retained for some of the side chains that are in contact with Trp24 in the native structure. Thus the distances Trp24–Phe137 and Trp24–Arg139 are 3.0–6.8 A ˚ and 2.7–8.4 A ˚ , respectively, in the group 3 conformers (Fig. 4). Mutational studies have shown that Trp24, and its side chain interactions, play an important role in stabilizing the RBP structure, and potentially in pre- venting misfolding [25]. Trp24 and Phe137 are in a hydrophobic cluster in the native structure, while the side chains of Trp24 and Arg139 form an amine– aromatic interaction that closes the base of the b-barrel structure. These residues are part of an evolutionary conserved set of residues in the lipocalin superfamily, which it is suggested may fold on a faster timescale than nonconserved regions [15]. In accord with this, stopped flow fluorescence studies have shown that Trp24 is in a near native-like hydrophobic environment Fig. 4. Normalized distributions of the distances between selected residues for the simulation conformers in groups 2 (top panels) and 3 (bottom panels). The shortest distances between atoms in these side chains in the native state structure of RBP are indicated in paren- theses. (A) Met73–Tyr90 (native state 3.8 A ˚ ); (B) Phe36–Tyr90 (native state 6.9 A ˚ ); (C) Phe20–Phe137 (native state 5.0 A ˚ ); (D) Phe36–Tyr133 (native state 3.4 A ˚ ); (E) Trp24–Phe137 (native 3.2 A ˚ ); (F) Trp24–Arg139 (native state 3.7 A ˚ ).The scale on the y -axis is arbitrary but the same for each pair of residues. E. Paci et al. MD simulations of the RBP molten globule state FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS 4831 during the early stages of folding [15]. The persist- ence of native-like contacts for Trp24 in the simula- tion group 3 conformers suggests that these contacts may be retained in the molten globule state. This would be consistent with the significance of this side chain, and its contacts, for the folding and stability of the protein. Common interactions may be topological constraints in the molten globule The simulation conformers in groups 1–3 all share the same topology, while varying to a large degree in sec- ondary structure persistence and side chain disorder. This study provides an ideal model system to investi- gate whether there is a common set of native-like inter- actions maintained within low energy partly unfolded structures, which provide a putative model for the molten globule. For this purpose, we computed the pairwise effective energy between pairs of residues, averaged over all the structures belonging to the differ- ent groups defined above. A network of the interac- tions was built, by linking together residues whose pairwise interaction is larger than a threshold (2.5 kcalÆ mol )1 ), and removing isolated residues. The resulting network is shown in Fig. 5 for the very native-like group 1 and for the molten globule-like group 3 struc- tures. The networks were analyzed using a network principle termed ‘betweenness-centrality’ [37,38]. This measure allows the identification of key nodes govern- ing the network of interactions in proteins [39,40]. The light grey residues in Fig. 5 have the highest between- ness score, and are therefore the most influential to the network. The interaction network representing group 3 is simpler, and the set of key nodes is smaller than for group 1. Interestingly, the structure of the networks of the group 1 and group 3 conformers is similar, and the set of residues that have a high betweenness in group 3 have also high betweenness in group 1 conformers. These are Arg10, Lys12, Asn14, Val107, Glu108, Thr109, Tyr111, Val116, Arg139 and Arg155. All these 10 residues are clustered together on the second face of the 1 b-barrel, in the same overall region of the tertiary structure. However, they reside in six different struc- tural elements, being located in the N-terminal region, on 2 b-strands F, G, H, in the F–G loop and in the C-terminal helix in the native RBP structure. There are wide variations in the characteristics of the contacts between these residues. For example, in some cases there are interactions between charged or polar side chain groups (e.g. Glu108 has contacts to Lys12 and R2 F45 S46 V42 A43 L35 A26 I41 E44 E16 G22 A130 S134 F135 E131 C174 A71 S21 R19 T128 A28 T56 R62 A55 A57 M53 S54 E33 G51 E39 G92 L37 S7 V6 V61 G59 L125 E49 L122 K85 M73 Y114 E72 E102 V74 G75 C4 E103 N124 W91 V93 K99 G100 W105 F86 T76 A84 T80 Y173 H170 G172 Q149 F77 C120 F137 L144 R166 R163 I106 Y165 A115 V69 E158 R153 K58 K17 Q156 Y118 K30 S132 K89 M88 T78 N101 K87 Q98 Q117 E68 K29 N40 N171 W24 V136 G127 Y90 H104 S119 Q38 R121 Y133 R60 M27 I168 R5 Y25 S138 T113 R10 R155 E140 E112 V107 V116 E108 K12 T109 N14 R139 Y111 R2 Q98 E102 N101 I106 C4 A115 Y114 Y90 K85 N124 V69 M73 A71 G75 E82 F77 S119 L125 G59 E158 N171 Q164 G172 Q149 Y173 R153 L144 T128 C129 F135 S134 V136 E140 F137 N66 C174 M27 E16 K17 E13 F36 S7 R19 E33 V6 E31 Y25 W24 S21 G22 Q38 E39 M53 S54 A55 A57 S46 F45 E44 V42 A43 T56 K58 K87 E68 V61 N40 K29 R60 R121 Y165 Q117 Y133 R166 K30 W105 S138 G127 E131 Y118 L122 A130 R163 S132 T113 R5 R139 R10 Y111 R155 V107 N14 V116 E108 T109 K12 A B Fig. 5. Network of effective interactions averaged over the structures of group 1 (A) and group 3 (B). The average effective energy between pairs of residues including solvation free energy was computed as described in Paci et al. [55]. The network was built connecting pairs of residues, whose absolute value of the interaction is larger than 2.5 kcalÆmol )1 , and that are more than four residues apart in the sequence. The threshold of 2.5 kcalÆmol )1 is somewhat arbitrary and is chosen so that the representation of the network of interactions is shown most clearly. Residues represented as light grey ovals are those with a betweenness larger than 5% (graphical representation and betweenness obtained with the program VISONE [56]). MD simulations of the RBP molten globule state E. Paci et al. 4832 FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS Arg155) while in other cases there are interactions between pairs of hydrophobic groups (e.g. Val107 has contacts to Val116). In addition, six of these 10 resi- dues are also among the most highly conserved resi- dues and interactions in the lipocalin superfamily (N14, V107, E108, T109, Y111, R139) [15]. The simi- larity of the results for the group 1 and group 3 con- formers suggests that many, but not all, of the interactions that are crucial for determining the native state fold of the protein are retained in the RBP mol- ten globule. Furthermore, the conservation of the resi- dues with the high betweenness in RBP across the lipocalin superfamily indicates the important role that folding to a specific native state plays in determining evolutionary selection. The results for RBP are partic- ularly interesting in the light of a recently reported and complementary analysis of networks of conserved interactions in the intracellular lipid-binding protein family [41]. Proteins in this family are similar to the lipocalins having a b-barrel structure. However, the proteins are in general smaller than the lipocalins and their fold is different, consisting of a 10-stranded b-barrel with a helix-turn-helix motif near the N-termi- nus. From this work on the intracellular lipid-binding protein family it was suggested that a network of con- served hydrophobic side chain interactions in the b-sheet region of the protein could provide a potential folding nucleation site. This has interesting parallels with an earlier reported study of the lipocalins [15]. Retinol binding site Retinol binds to RBP in the core of the b-barrel. When bound, retinol is almost totally encapsulated by the protein, the b-ionone ring lying in the centre of the protein with the isoprene chromophore stretching along the barrel axis [35]. RBP releases retinol under low pH conditions, undergoing the transition to the molten globule state. The changes to the structure seen in the group 3 conformers, especially the disruption in the strand E to F region, lead to a significant opening of the retinol binding site to solvent, and so would prompt loss of bound retinol in the molten globule state. The simulation analysis therefore proposes that changes to the E to F region play an important role in allowing retinol release from RBP. This proposal is supported by data from studies of ligand binding to a number of b-lactoglobulins, other members of the lipocalin superfamily [42,43]. Here, an opening and closing of the E-F loop region has been shown to pro- vide a mechanism for the binding and release of lig- ands. In the b-lactoglobulins the changes are localized in the E–F loop, and are prompted by the protonation of a glutamate side chain within the loop. In RBP, the simulation results suggest that the disorder in the E to F region is part of much more wide spread conforma- tional changes, as the protein undergoes the transition into the molten globule state. Prior to the simulations reported here, retinol was removed from the X-ray structure coordinates. This gave an increase of 251 A ˚ 2 in the solvent accessible surface area of the protein. Comparisons of the side chain solvent accessibility of the X-ray structure of RBP (without retinol bound) with that of the group 3 conformers show that, on average, there is a further increase in mean accessibility of 6.8 A ˚ 2 per residue for the group 3 conformers. Many of the side chains that make contacts with retinol when it is bound to the native state have, however, much larger increases in mean side chain accessibility in the group 3 conform- ers. For example, for Leu63, Met73 and Phe77 the mean side chain accessibility increases by 59.3, 85.6 and 77.9 A ˚ 2 , respectively (Fig. 6). Experimentally the molten globule state of RBP has been shown to bind strongly the hydrophobic dye 8-anilino-1-napthalene- sulphonate (ANS), giving an intense fluorescence spec- trum [24]. This property, characteristic of molten globules, is generally recognized to reflect the presence of exposed hydrophobic surfaces. The simulation group 3 conformers show that in the case of RBP the exposed hydrophobic side chain groups from the reti- nol binding site are likely to be responsible, at least in part, for the observed ANS binding. Transthyretin binding site RBP circulates in the blood stream in a one-to-one complex with a second serum protein, transthyretin (TTR) [32]. This large complex prevents the smaller RBP from being filtered through the renal glomeruli [19]. Upon reaching target tissues, RBP and trans- thyretin dissociate, and retinol is released for delivery to the cells, in a yet undetermined mechanism. Four regions of the sequence of RBP at the opening of the barrel make contacts with TTR [32]. The residues in RBP in three of the regions involved in these contacts are identified in Fig. 6. The protein C-terminus is the fourth region of RBP which makes contacts with TTR. These residues were missing from the X-ray structure of RBP [35], and so were excluded from the simulations reported here. The binding of TTR to RBP has been shown to stabilize the binding of retinol to RBP. Studies of the interactions of chimaera of RBP and epididymal retinoic acid binding pro- tein (ERABP) with TTR have identified the region of the RBP sequence that is key to providing this E. Paci et al. MD simulations of the RBP molten globule state FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS 4833 stabilization [33]. The residues concerned are those in the E-F loop, where the most major changes in struc- ture are observed in the group 3 simulation conform- ers. This suggests that the dissociation of the RBP– TTR complex may be linked to release of retinol bound to RBP, via conformational changes in the E–F region of the protein. In this respect it is particularly interesting that, in some of the simulation conformers analyzed, residues in the E-F region adopted helical turns. It is therefore possible that a conversion in sec- ondary structure could be involved in the RBP–TTR dissociation mechanism. Further work is needed, how- ever, to investigate this mechanism in more detail, including a study of the role of the C-terminal region of the RBP sequence which was excluded from the simulations reported here. Comparison with other molten globule states Overall, analysis of the partially folded conformers generated for RBP, and comparison with experimental data, indicates that the structures in group 3 can pro- vide a model for the ensemble of conformers popu- lated in the molten globule state of the protein. These conformers retain considerable secondary structure, but show disorder in side chain packing, and have exposed hydrophobic groups. In the group 3 conform- ational ensemble the central strands of the two ortho- gonal b-sheets show a high persistence. The structure in the outer strands is more fluctuating in nature. It is interesting to compare these secondary structure char- acteristics with those seen for the molten globule states of other proteins that have been studied in detail experimentally. Many of the proteins whose molten globules have been characterized have predominantly a-helical secondary structure in their native state, and this is largely retained in the molten globule state. Moreover for proteins that contain both a-helices and b-strands in the native state, it is the a-helical part of the structure that particularly persists in the molten globule. The b-sheet regions are more unstructured, in contrast to the results for RBP. This is seen, for exam- ple, in human a-lactalbumin [12] and Escherichia coli ribonuclease HI [13]. Comparison with data for the molten globule state of two proteins which have a native state fold that is rich in b-sheet secondary struc- ture, carbonic anhydrase and b-lactoglobulin, is there- fore particularly relevant. Human carbonic anhydrase II contains 10 b-strands in the native state, but differs topologically from RBP, having an ab roll fold [44]. In the partially folded state the central strands 3–7 have native-like structure, but less ordered structure is found in the peripheral strands [45,46]. Hence, as in the case of RBP, the core part of -100 -50 0 50 Surf-Surf X TTR binding residues 0 10203040 50 60 70 80 90 100 110 120 130 140 150 160 170 residue number -100 -50 0 50 Group2 Group3 Fig. 6. Difference between the average side chain accessibility of each residue in the group 2 (upper panel) or group 3 (lower panel) simula- tion conformers and the side chain accessibility in the X-ray structure of native RBP (with retinol removed). Black bars indicate the residues that make contacts to bound retinol in the native state, and triangles show the residues that are involved in binding to TTR. The solvent accessible surface area of the side chains has been calculated using the program NACCESS [57]. The average increase in surface accessible area per residues is 6 and 7 A ˚ 2 in the structures belonging to group 2 and group 3, respectively, compared to a change of )0.5 A ˚ 2 in the equilibration (control) simulation. MD simulations of the RBP molten globule state E. Paci et al. 4834 FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS the b-sheet secondary structure is retained in the mol- ten globule state, while external regions of the struc- ture are more disordered. b-lactoglobulin is a member of the lipocalin superfamily, and like RBP its native structure contains an up-and-down b-barrel and one C-terminal a-helix [42]. Hydrogen exchange studies of the molten globule state of equine b-lactoglobulin show that the high protection factors are seen for resi- dues in strands G and H (values > 100 for five resi- dues). These strands are linked by a disulphide bridge between Cys106 and Cys119. Moderate protection fac- tors (10–20) are seen for some residues in the regions corresponding to strands A, D and F in the native state, and also the a-helix. Residues out of these regions have protection factors less than 10 (with the exception of Asn53 in the B–C loop which has a pro- tection factor of 11) [47]. These data are consistent with a high persistence for native-like secondary struc- ture in the central parts of the second b-sheet (residues in strands F, G and H together with part of strand A), while some secondary structure of a less persistent nat- ure in the centre of the first b-sheet (particularly for strand D) and in the a-helix [47]. Results for bovine b-lactoglobulin are very similar, with a high level of hydrogen exchange protection being seen in the central parts of the second b-sheet (residues in strands F, G, H and A) in the partially folded state formed at pH 2 [48]. In addition, for bovine b-lactoglobulin significant hydrogen exchange protection persists for residues in b-strands G and H in the cold denatured state [49]. These data for b-lactoglobulin are similar to the results reported here, although in the RBP simulation con- formers there is an equivalent level of persistence in the central regions of both of the two b-sheets, while in b-lactoglobulin the second sheet predominates. The proposed similarity between the molten globule states of RBP and b-lactoglobulin suggests that the partially folded protein characteristics identified here may be typical of those adopted by other proteins in the lipo- calin superfamily. Conclusions In the model for the molten globule state of RBP reported here the protein retains a persistent b-sheet core, although even in these regions there is consider- able variation in the side chain packing. Out of the b-sheet core there is much more disorder across the conformational ensemble. The network analysis of the simulation conformers shows that there is a small subset of persistent key interactions. The most signifi- cant changes in the structure are seen in the region extending from the start of b-strand E to the middle of b-strand F (residues 85–104). It is possible that the conformational changes in this region of the protein in the simulation conformers, including the presence of some a-helical character, could play an important role in the mechanism for the dissociation of the TTR– RBP complex and retinol release at the cell surface. Moreover, similarities between the simulation con- formers of RBP and experimental data for b-lactoglob- ulin, suggest that the model for the molten globule of RBP reported here may have relevance to our under- standing of the properties and ligand binding of other members of the lipocalin superfamily. This is of partic- ular interest in the light of the development of engineered lipocalins for carrying novel ligands in therapeutic approaches [23,50,51]. Experimental procedures The initial structure used in the simulations was the crystal structure of human serum retinol-binding protein at 2 A ˚ resolution (PDB entry 1RBP [35], Fig. 2), with the bound retinol ligand removed. The C-terminal region of the RBP sequence is missing from the X-ray structure, as these resi- dues could not be located in the electron density map (resi- dues 175–182). We therefore also chose to exclude these eight C-terminal residues from the simulations reported here, although it is possible that this truncation of the sequence could affect the stability of non-native states of the protein. An equilibration (control) simulation was run for 2 ns at 300 K. The exploration of the conformational space outside the native state at room temperature was car- ried out by a biased molecular dynamics (BMD) scheme, similar to that used by Paci et al. [30]. All simulations were performed in implicit solvent (EEF1 [52]). The system was initially energy minimized to remove bad contacts, heated up to 300 K in 0.6 ns and then equilibrated for 2 ns. Dur- ing the last 1 ns of this equilibration simulation the average RMSD from the native state crystal structure was 2.4 (3.2) A ˚ for Ca (all atoms): i.e. the native state is relat- ively stable with the implicit solvation method employed. Following this, a 6 ns simulation was performed using biased molecular dynamics, with a perturbation increasing the protein radius of gyration. During this simulation, the radius of gyration and solvent accessible surface area of the protein increased up to 30% relative to that of the native structure, while the heavy atom RMSD from the native structure reached 13 A ˚ . Five conformations were picked out from along the tra- jectory, at approximately equal intervals in radius of gyra- tion value, ranging from one with an almost native-like radius of gyration to a maximally denatured conformer. Five 1.6-ns simulations, one for each initial configuration, were started with a perturbation favouring the loss of native contacts between side-chain heavy atoms. A configuration E. Paci et al. MD simulations of the RBP molten globule state FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS 4835 [...]... to the adiabatic bias used to induce the loss of native features, the energy of the conformers is no more than 20 kcalÆmol)1 larger than the average energy in the native state) These conformations span a range between 2.1 and ˚ 11.9 A in heavy atom RMSD from the native X-ray struc˚ ture and between 15.7 and 19.1 A in protein radius of gyration From each of these 160 conformations a 1.2 ns simulation. .. alpha-lactalbumin by molecular dynamics simulation J Mol Biol 306, 329–347 Marchi M & Ballone P (1999) Adiabatic bias molecular dynamics: a method to navigate the conformational space of complex molecular systems J Chem Phys 110, 3697–3702 Naylor HM & Newcomer ME (1999) The structure of human retinol-binding protein (RBP) with its carrier protein transthyretin reveals an interaction with the carboxy... (Nall BT & Dill KA, eds), pp 175–181 AAAS Publications, Washington DC Day R & Daggett V (2003) All-atom simulations of protein folding and unfolding Adv Protein Chem 66, 373–403 Smith LJ (2004) Computational methods for generating models of denatured and partially folded proteins Methods 34, 144–150 Paci E, Smith LJ, Dobson CM & Karplus M (2001) Exploration of partially unfolded states of human alpha-lactalbumin... for access to its facilities Some of the simulations reported in this paper were performed while EP was at the Department of Biochemistry, University of Zurich References 1 Ptitsyn OB (1995) Molten globule and protein folding Adv Protein Chem 47, 83–229 2 Arai M & Kuwajima K (2000) Role of the molten globule state in protein folding Adv Protein Chem 53, 209–282 3 Khurana R, Gillespie JR, Talapatra A, ... Redfield C (2004) Using nuclear magnetic resonance spectroscopy to study molten globule states of proteins Methods 34, 121–132 Vendruscolo M, Paci E, Karplus M & Dobson CM (2003) Structures and relative free energies of partially folded states of proteins Proc Natl Acad Sci USA 100, 14817–14821 Ohgushi M & Wada A (1983) Molten- globule state – a compact form of globular-proteins with mobile sidechains FEBS... simulation was started without any bias (the first 0.2 ns were disregarded in the following analysis) We thus produced 160 ns of unperturbed equilibrium trajectories spanning the conformational space starting from 160 diverse initial configurations The total length of the simulation was more than 300 ns The present approach, while not suitable to determine the free energy surface of the protein, which... LJ, Ionescu-Zanetti C, Millett I & Fink AL (2001) Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates Biochemistry 40, 3525–3535 4 Dobson CM (2001) The structural basis of protein folding and its links with human disease Philos Trans Roy Soc London B 356, 133–145 5 Kuwajima K (1996) The molten globule state of alphalactalbumin FASEB J 10, 102–109...MD simulations of the RBP molten globule state every 0.4 ns was saved, and used as an initial conformation to perform eight sequential 0.4 ns simulations with a perturbation increasing the RMSD from the initial structure This procedure generated, in a relatively short simulation time (6+72 ns), 160 distinct conformations (coordinates and velocities) that are independent and close to equilibrium... hydrogen exchange J Mol Biol 299, 757–770 Ragona L, Pusterla F, Zetta L, Monaco HL & Molinari H (1997) Identification of a conserved hydrophobic cluster in partially folded bovine b-lactoglobulin at pH 2 Folding Design 2, 281–290 4837 MD simulations of the RBP molten globule state 49 Katou H, Hoshino M, Kamikubo H, Batt CA & Goto Y (2001) Native-like beta-hairpin retained in the colddenatured state of bovine... beta-lactoglobulin J Mol Biol 310, 471–484 50 Beste G, Schmidt FS, Stibora T & Skerra A (1999) Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold Proc Natl Acad Sci USA 96, 1898–1903 51 Schlehuber S & Skerra A (2002) Tuning ligand affinity, specificity, and folding stability of an engineered lipocalin variant – a so-called ‘anticalin’ – using a molecular random . Characterization of the molten globule state of retinol-binding protein using a molecular dynamics simulation approach Emanuele Paci 1 , Lesley. conformers taken at 10-ps intervals along the unbiased simulations. The radius of gyration and RMSD values are calculated using all heavy atoms. The black diamond