A systematic study of the energetics involved in structural changes upon association and connectivity in protein interaction networks

The study of protein binding mechanisms is a major topic of research in structural biology. Here, we implement a combination of metrics to systematically assess the cost of backbone conformational changes that protein domains undergo upon association. Through the analyses of 2090 unique unbound → bound transitions, from over 12,000 structures, we show that two-thirds of these proteins do not suffer significant structural changes upon binding, and could thus fit the lock-and-key model well. Among the remaining proteins, one-third explores the bound conformation in the unbound state (conformational selection model) and, while most transitions are possible from an energetic perspective, a few do require external help to break the thermodynamic barrier (induced fit model). We also analyze the relationship between conformational transitions and protein connectivity, finding that, in general, domains interacting with many partners undergo smaller changes upon association, and are less likely to freely explore larger conformational changes.     

Probing Structural Changes during Self-assembly of Surface-Active Hydrophobin Proteins that Form Functional Amyloids in Fungi

Hydrophobins are amphiphilic proteins secreted by filamentous fungi in a soluble form, which can self-assemble at hydrophilic/hydrophobic or water/air interfaces to form amphiphilic layers that have multiple biological roles. We have investigated the conformational changes that occur upon self-assembly of six hydrophobins that form functional amyloid fibrils with a rodlet morphology. These hydrophobins are present in the cell wall of spores from different fungal species. From available structures and NMR chemical shifts, we established the secondary structures of the monomeric forms of these proteins and monitored their conformational changes upon amyloid rodlet formation or thermal transitions using synchrotron radiation circular dichroism and Fourier-transform infrared spectroscopy (FT-IR). Thermal transitions were followed by synchrotron radiation circular dichroism in quartz cells that allowed for microbubbles and hence water/air interfaces to form and showed irreversible conformations that differed from the rodlet state for most of the proteins. In contrast, thermal transitions on hermetic calcium fluoride cells showed reversible conformational changes. Heating hydrophobin solutions with a water/air interface on a silicon crystal surface in FT-IR experiments resulted in a gain in β-sheet content typical of amyloid fibrils for all except one protein. Rodlet formation was further confirmed by electron microscopy. FT-IR spectra of pre-formed hydrophobin rodlet preparations also showed a gain in β-sheet characteristic of the amyloid cross-β structure. Our results indicate that hydrophobins are capable of significant conformational plasticity and the nature of the assemblies formed by these surface-active proteins is highly dependent on the interface at which self-assembly takes place.     

Conformational sampling and nucleotide-dependent transitions of the GroEL subunit probed by unbiased molecular dynamics simulations

GroEL is an ATP dependent molecular chaperone that promotes the folding of a large number of substrate proteins in E. coli. Large-scale conformational transitions occurring during the reaction cycle have been characterized from extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in the allosteric response to ATP and the nucleotide-driven reaction cycle are not completely established. Here we describe extensive (in total long) unbiased molecular dynamics (MD) simulations that probe the response of GroEL subunits to ATP binding. We observe nucleotide dependent conformational transitions, and show with multiple 100 ns long simulations that the ligand-induced shift in the conformational populations are intrinsically coded in the structure-dynamics relationship of the protein subunit. Thus, these simulations reveal a stabilization of the equatorial domain upon nucleotide binding and a concomitant "opening" of the subunit, which reaches a conformation close to that observed in the crystal structure of the subunits within the ADP-bound oligomer. Moreover, we identify changes in a set of unique intrasubunit interactions potentially important for the conformational transition.     

Tracking Molecular Interactions in Membranes by Simultaneous ATR-FTIR-AFM

In situ atomic force microscopy (AFM) is an exceedingly powerful and useful technique for characterizing the structure and assembly of proteins in real-time, in situ, and especially at model membrane interfaces, such as supported planar lipid bilayers. There remains, however, a fundamental challenge with AFM-based imaging. Conclusions are inferred based on morphological or topographical features. It is conventionally very difficult to use AFM to confirm specific molecular conformation, especially in the case of protein-membrane interactions. In this case, a protein may undergo subtle conformational changes upon insertion in the membrane that may be critical to its function. AFM lacks the ability to directly measure such conformational changes and can, arguably, only resolve features that are topographically distinct. To address these issues, we have developed a platform that integrates in situ AFM with attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. This combination of tools provides a unique means of tracking, simultaneously, conformational changes, not resolvable by in situ AFM, with topographical details that are not readily identified by conventional spectroscopy. Preliminary studies of thermal transitions in supported lipid bilayers and direct evidence of lipid-induced conformational changes in adsorbed proteins illustrates the potential of this coupled in situ functional imaging strategy.     

Conformational changes and association of chemically modified chymotrypsins

The low pH conformational transitions of a series of modified chymotrypsins have been examined and compared to the association properties of these same proteins in order to determine if the effect of the modification was directly upon the association or indirectly through conformational changes. The modifications of α-chymotrypsin that have been studied are serine-195 modified with acetyl or with phenylmethane sulfonyl groups, His-57 modified by tosylamidophenylethyl chloromethyl ketone or with a methyl group, and Met-192 modified with a nitrophenacyl group. Chymotrypsinogen and native and modified delta-chymotrypsin have also been studied. Ultraviolet difference spectra and optical rotatory dispersion measurements show that the various proteins may be divided into three groups depending upon their response in the low pH transition. These groupings based upon conformational transitions are not divided in the same manner as were those based upon the effect of the modifications on the association process itself. The latter process had earlier been found to correlate with the size of the modification in the active site. From these findings it has been concluded that the effect of the modifications on the association was directly upon the interacting subunit interface and that the observed conformational change is only incidental to this effect. Therefore, the amino acids involved in the association in solution are the same as those involved in the dimerization in the crystal, namely, the active site region of Ser-195, His-57, and Met-192. This is the most direct demonstration that the mode of association of a protein in solution is the same as the mode of association in the crystal.     

Side-chain flexibility in protein-ligand binding: the minimal rotation hypothesis

The goal of this work is to learn from nature about the magnitudes of side-chain motions that occur when proteins bind small organic molecules, and model these motions to improve the prediction of protein-ligand complexes. Following analysis of protein side-chain motions upon ligand binding in 63 complexes, we tested the ability of the docking tool SLIDE to model these motions without being restricted to rotameric transitions or deciding which side chains should be considered as flexible. The model tested is that side-chain conformational changes involving more atoms or larger rotations are likely to be more costly and less prevalent than small motions due to energy barriers between rotamers and the potential of large motions to cause new steric clashes. Accordingly, SLIDE adjusts the protein and ligand side groups as little as necessary to achieve steric complementarity. We tested the hypothesis that small motions are sufficient to achieve good dockings using 63 ligands and the apo structures of 20 different proteins and compared SLIDE side-chain rotations to those experimentally observed. None of these proteins undergoes major main-chain conformational change upon ligand binding, ensuring that side-chain flexibility modeling is not required to compensate for main-chain motions. Although more frugal in the number of side-chain rotations performed, this model substantially mimics the experimentally observed motions. Most side chains do not shift to a new rotamer, and small motions are both necessary and sufficient to predict the correct binding orientation and most protein-ligand interactions for the 20 proteins analyzed.     

Surface plasmon resonance measurement of pH-induced responses of immobilized biomolecules: conformational change or electrostatic interaction effects?

Recently, the observation of pH-induced conformational changes of biomolecules supported on carboxymethyldextran (CMD)-coated surfaces measured using surface plasmon resonance (SPR) has been reported. However, it is apparent that the evidence reported in the literature is ambiguous. The research presented in this paper describes investigations to study the changing SPR signal of immobilized biomolecules as a function of varying pH, to provide a detailed understanding of the origin of the pH-induced changes in the SPR profile. SPR measurements were performed with cytochrome c, concanavalin A, and poly-L-lysine, biomolecules that exhibit diverse conformational responses to changing pH, covalently immobilized onto CMD-coated supports. These SPR measurements were supported by circular dichroism (CD) solution studies. The SPR profiles recorded were not consistent with the conformational transitions of the biomolecules as observed using CD. An alternative explanation for the observed shifts in SPR is proposed, which explains the SPR profiles in terms of electrostatic interaction effects between the immobilized biomolecules and the carboxymethyldextran matrix.     

Alcohol-induced conformational changes of ubiquitin

Ubiquitin has been found to be soluble in ethylene glycol and alcohols as the perchlorate or hydrochloride salt. When the effect of alcohol on the structure of ubiquitin is examined, two reversible conformational transitions are observed. Upon lowering the dielectric constant of aqueous alcohol solutions of ubiquitin from 80 to 45, the native structure of ubiquitin is converted to a form consistent with 50% helical structure. This conformational change results in a change in exposure to solvent of the single methionine and the single tyrosine residues of ubiquitin. In agreement with crystallographic results, these residues are buried in the native conformation but become fully exposed to solvent upon undergoing this transition. Further lowering of the dielectric constant to 20 results in the accumulation of a conformation with almost complete helical structure. Thus, hydrophobic interactions cause facile conformational changes in the ubiquitin structure. These results are discussed in terms of a preferential solvation model. It is shown that the results obtained with different alcohols can be normalized by the use of a dielectric constant scale. This normalization corrects for the different molar volumes of different alcohols, allows comparison of results obtained with different alcohols, and should be useful in studying this phenomenon with different proteins.     

Thiol-based redox signalling: rust never sleeps

Cysteine residues in proteins are covalently modified under conditions of oxidative and nitrosative stress by oxidation, nitrosation, glutathionylation and disulfide formation. Modifications induce conformational changes in substrate proteins, effecting signal cascades that evoke a biological response. A growing number of structures with modified cysteines are allowing a piecemeal understanding of the mechanistic aspects of these signalling pathways to emerge. Conformational changes upon conjugation of nitric oxide and glutathione are generally small and often accompanied by a local increase in protein disorder. Burial of nitric oxide is also apparent, which may increase the timeframe of signalling. Conformational changes upon disulfide formation/reduction range from the small to the spectacular. They include order/disorder transitions; oxidation of disulfides following expulsion of metals such as Zn; major reorganisation or "morphing" of portions of the polypeptide backbone; and changes in quaternary structure including domain swapping.     

Protein structural changes induced by their uptake at interfaces

For insertion into lipidic media, most hydrosoluble proteins must cross the lipid-water interface and thus undergo conformational transitions. According to their chemical sequences these transitions may be restricted to changes involving only the tertiary structure, while for other proteins this environment modification will induce drastic changes such as the unfolding of large domains. The structural transitions are mainly governed by the presence of hydrophobic domains and/or by the existence of induced amphipathic properties.     

Effect of sucrose and maltodextrin on the physical properties and survival of air-dried Lactobacillus bulgaricus: an in situ fourier transform infrared spectroscopy study

The effect of sucrose, maltodextrin and skim milk on survival of L. bulgaricus after drying was studied. Survival could be improved from 0.01% for cells that were dried in the absence of protectants to 7.8% for cells dried in a mixture of sucrose and maltodextrin. Fourier transform infrared spectroscopy (FTIR) was used to study the effect of the protectants on the overall protein secondary structure and thermophysical properties of the dried cells. Sucrose, maltodextrin and skim milk were found to have minor effects on the membrane phase behavior and the overall protein secondary structure of the dried cells. FTIR was also used to show that the air-dried cell/protectant solutions formed a glassy state at ambient temperature. 1-Palmitoyl 2-oleoyl phosphatidyl choline (POPC) was used in order to determine if sucrose and maltodextrin have the ability to interact with phospholipids during drying. In addition, the glass transition temperature and strength of hydrogen bonds in the glassy state were studied using this model system. Studies using poly-L-lysine were done in order to determine if sucrose and maltodextrin are able to stabilize protein structure during drying. As expected, sucrose depressed the membrane phase transition temperature (Tm) of POPC in the dried state and prevented conformational changes of poly-L-lysine during drying. Maltodextrin, however, did not depress the Tm of dried POPC and was less effective in preventing conformational changes of poly-L-lysine during drying. We suggest that when cells are dried in the presence of sucrose and maltodextrin, sucrose functions by directly interacting with biomolecules, whereas maltodextrin functions as an osmotically inactive bulking compound causing spacing of the cells and strengthening of the glassy matrix.     

Nucleic acid actions on abnormal protein aggregation,phase transitions and phase separation

Liquid–liquid phase separation (LLPS) and phase transitions (PT) of proteins, which include the formation of gel- and solid-like species, have been characterized as physical processes related to the pathology of conformational diseases. Nucleic acid (NA)-binding proteins related to neurodegenerative disorders and cancer were shown by us and others to experience PT modulated by different NAs. Herein, we discuss recent work on phase separation and phase transitions of two amyloidogenic proteins, i.e. the prion protein (PrP) and p53, which undergo conformational changes and aggregate upon NA interaction. The role of different NAs in these processes is discussed to shed light on the relevance of PSs and PTs for both the functional and pathological roles of these mammalian proteins.     

Side-chain conformational changes upon Protein-Protein Association

Conformational changes upon protein-protein association are the key element of the binding mechanism. The study presents a systematic large-scale analysis of such conformational changes in the side chains. The results indicate that short and long side chains have different propensities for the conformational changes. Long side chains with three or more dihedral angles are often subject to large conformational transition. Shorter residues with one or two dihedral angles typically undergo local conformational changes not leading to a conformational transition. A relationship between the local readjustments and the equilibrium fluctuations of a side chain around its unbound conformation is suggested. Most of the side chains undergo larger changes in the dihedral angle most distant from the backbone. The frequencies of the core-to-surface interface transitions of six nonpolar residues and Tyr are larger than the frequencies of the opposite surface-to-core transitions. The binding increases both polar and nonpolar interface areas. However, the increase of the nonpolar area is larger for all considered classes of protein complexes, suggesting that the protein association perturbs the unbound interfaces to increase the hydrophobic contribution to the binding free energy. To test modeling approaches to side-chain flexibility in protein docking, conformational changes in the X-ray set were compared with those in the docking decoy sets. The results lead to a better understanding of the conformational changes in proteins and suggest directions for efficient conformational sampling in docking protocols.     

The decisive role of the water structure in changes of conformation of nucleic acids.

This review summarizes data on the structure and properties of water under normal conditions, at high salt concentration and under high pressure. We correlate the observed conformational transitions in nucleic acids with changes in water structure and activity, and suggest a mechanism of conformational transitions of nucleic acid involving these changes. We conclude that the Z-DNA form is induced only at low water activity caused by high salt concentrations and/or high pressure.     

Compressibility of protein transitions

We review the results of compressibility studies on proteins and low molecular weight compounds that model the hydration properties of these biopolymers. In particular, we present an analysis of compressibility changes accompanying conformational transitions of globular proteins. This analysis, in conjunction with experimental compressibility data on protein transitions, were used to define the changes in the hydration properties and intrinsic packing associated with native-to-molten globule, native-to-partially unfolded, and native-to-fully unfolded transitions of globular proteins. In addition, we discuss the molecular origins of predominantly positive changes in compressibility observed for pressure-induced denaturation transitions of globular proteins. Throughout this review, we emphasize the importance of compressibility data for characterizing protein transitions, while also describing how such data can be interpreted to gain insight into role that hydration and intrinsic packing play in modulating the stability of and recognition between proteins and other biologically important compounds.     

Comparative molecular dynamics—Similar folds and similar motions?

Proteins possessing the same fold may undergo similar motions, particularly if these motions involve large conformational transitions. The increasing amounts of structural data provide a useful starting point with which to test this hypothesis. We have performed a total of 0.29 micros of molecular dynamics across a series of proteins within the same fold family (periplasmic binding proteinlike) in order to address to what extent similarity of motion exists. Analysis of the local conformational space on these timescales (10-20 ns) revealed that the behavior of the proteins could be readily distinguished between an apo-state and a ligand-bound state. Moreover, analysis of the root-mean-square fluctuations reveals that the presence of the ligand exerts a stabilizing effect on the protein, with similar motions occurring, but with reduced magnitude. Furthermore, the conformational space in the presence of the ligand appears to be dictated by sequence but not by the type of ligand present. In contrast, apo-simulations showed considerable overlap of conformational space across the fold as a result of their ability to undergo larger fluctuations. Indeed, we observed several transitions from different simulations between states corresponding to the closed-cleft and open-cleft forms of the fold, with the predominant motions being conserved across the different proteins. Thus, large-scale conformational changes do indeed appear to be conserved across this fold architecture, but smaller conformational motions appear to reflect the differences in sequence and local fold.     

Ordered conformational change in the protein backbone: prediction of conformationally variable positions from sequence and low-resolution structural data

Ordered conformational changes are an important structural property of proteins and are involved in a variety of fundamental biological activities. Large-scale analyses of the implications of such changes for protein function and dysfunction require efficient methods for automated recognition of conformationally variable residue positions. The goal of this work was to study sequence and low-resolution structural properties of residue positions that change backbone conformation upon changes in protein environment and the utility of these properties for automated recognition of such conformationally variable positions. This study was performed using a large nonredundant set of experimentally characterized proteins that undergo ordered conformational transitions obtained from the Database of Macromolecular Movements. The results of this study show that ordered changes in backbone conformation are not limited to solvent accessible loop regions. A considerable fraction of conformationally variable positions is observed in helices and strands, and in buried positions. Conformationally variable positions are less conserved in evolution. Local patterns of (a) sequence neighbors, (b) evolutionary conservation, and (c) solvent accessibility can be used to predict conformationally variable positions with balanced sensitivity and specificity, albeit with large variance at the level of individual proteins. However, including a pattern of secondary structure into the prediction scheme results in a highly unbalanced performance when all conformationally variable positions located in regular secondary structure are misclassified. Application of the present methodology to the prion protein (PrP) shows that conformationally variable positions predicted in its ordered C-terminal domain are located within segments presumed to be involved in refolding of PrP.     

Conformational changes in the amino-terminal helix of the G protein alpha(i1) following dissociation from Gbetagamma subunit and activation

G protein alpha subunits mediate activation of signaling pathways through G protein-coupled receptors (GPCR) by virtue of GTP-dependent conformational rearrangements. It is known that regions of disorder in crystal structures can be indicative of conformational flexibility within a molecule, and there are several such regions in G protein alpha subunits. The amino-terminal 29 residues of Galpha are alpha-helical only in the heterotrimer, where they contact the side of Gbeta, but little is known about the conformation of this region in the active GTP bound state. To address the role of the Galpha amino-terminus in G-protein activation and to investigate whether this region undergoes activation-dependent conformational changes, a site-directed cysteine mutagenesis study was carried out. Engineered Galpha(i1) proteins were created by first removing six native reactive cysteines to yield a mutant Galpha(i1)-C3S-C66A-C214S-C305S-C325A-C351I that no longer reacts with cysteine-directed labels. Several cysteine substitutions along the amino-terminal region were then introduced. All mutant proteins were shown to be folded properly and functional. An environmentally sensitive probe, Lucifer yellow, linked to these sites showed a fluorescence change upon interaction with Gbetagamma and with activation by AlF(4)(-). Other fluorescent probes of varying charge, size, and hydrophobicity linked to amino-terminal residues also revealed changes upon activation with bulkier probes reporting larger changes. Site-directed spin-labeling studies showed that the N-terminus of the Galpha subunit is dynamically disordered in the GDP bound state, but adopts a structure consistent with an alpha-helix upon interaction with Gbetagamma. Interaction of the resulting spin-labeled Galphabetagamma with photoactivated rhodopsin, followed by rhodopsin-catalyzed GTPgammaS binding, caused the amino-terminal domain of Galpha to revert to a dynamically disordered state similar to that of the GDP-bound form. Together these results suggest conformational changes occur in the amino-termini of Galpha(i) proteins upon subunit dissociation and upon activating conformational changes. These solution studies reveal insights into conformational changes that occur dynamically in solution.     

血清白蛋白在多聚阳离子－壳聚糖共混材料表面的构象变化及其与细胞生长的关系

用L-多聚赖氨酸、聚乙烯亚胺及L-多聚鸟氨酸三种多聚阳离子对壳聚糖进行共混修饰,制备了三种共混材料.在这些材料表面吸附了血清白蛋白,并利用圆二色(CD)光谱研究了白蛋白吸附到材料表面后的构象变化.结果显示,与天然状态相比,白蛋白吸附到共混材料表面后,其α-螺旋、β-折叠及无规则卷曲的含量均发生了明显改变.通过研究MC3T3-E1细胞在这些材料表面的生长情况,发现细胞的增殖与血清白蛋白的构象变化有一定关系,在吸附的白蛋白构象与天然构象最接近的共混材料表面,MC3T3-E1细胞增殖水平最高.     

Substrate-dependent morphology of supramolecular assemblies: fibrillin and type-VI collagen microfibrils

Substrate hydrophobicity/hydrophilicity has previously been shown to affect the morphology and biological function of isolated proteins. We have employed atomic force microscopy to investigate substrate dependent morphologies of two biochemically distinct native supramolecular assemblies: fibrillin and type-VI collagen microfibrils. These morphologically heterogeneous microfibrillar systems are found in many vertebrate tissues where they perform structural and cell-signaling roles. Fibrillin microfibrils adsorbed to a hydrophilic mica substrate adopted a diffuse morphology. Fibrillin microfibrils adsorbed to mica coated with poly-L-lysine or to borosilicate glass substrates had a more compact morphology and a directional asymmetry to the bead, which was not present on mica alone. Intermediate morphologies were observed along a substrate gradient. The classical double-beaded appearance of type-VI collagen microfibrils was evident on mica coated with poly-L-lysine and on glass. On hydrophilic mica, morphology was severely disrupted and there was a major conformational reorganization along the whole collagen microfibril repeat. These observations of substrate dependent conformation have important implications for the interpretation of data from in vitro protein interaction assays and cellular signaling studies. Furthermore, conformational changes may be induced by local charge environments in vivo, revealing or hiding binding sites.