Limited conformational change of beta-lactoglobulin when adsorbed at the air-water interface

Detailed insight can be obtained from proteins at and near the air-water interface using external reflection IR and circular dichroism techniques. Besides information on local protein concentrations and surface layer thickness, it is shown that beta-lactoglobulin displays a limited unfolding at the interface. The conformational change is comparable to that observed upon heat-induced aggregation of the protein and can be understood in view of the high surface concentration of the protein (approximately 40% volume fraction). The layer thickness and the conformational properties of the protein do not depend on the bulk concentration. After adsorption of beta-lactoglobulin to a preformed lipid monomolecular layer a similar conformational change is induced, suggesting that the folding properties of the protein itself determine the extent of conformational changes at the interfaces.     

A Correspondence Between Solution-State Dynamics of an Individual Protein and the Sequence and Conformational Diversity of its Family

Conformational ensembles are increasingly recognized as a useful representation to describe fundamental relationships between protein structure, dynamics and function. Here we present an ensemble of ubiquitin in solution that is created by sampling conformational space without experimental information using “Backrub” motions inspired by alternative conformations observed in sub-Angstrom resolution crystal structures. Backrub-generated structures are then selected to produce an ensemble that optimizes agreement with nuclear magnetic resonance (NMR) Residual Dipolar Couplings (RDCs). Using this ensemble, we probe two proposed relationships between properties of protein ensembles: (i) a link between native-state dynamics and the conformational heterogeneity observed in crystal structures, and (ii) a relation between dynamics of an individual protein and the conformational variability explored by its natural family. We show that the Backrub motional mechanism can simultaneously explore protein native-state dynamics measured by RDCs, encompass the conformational variability present in ubiquitin complex structures and facilitate sampling of conformational and sequence variability matching those occurring in the ubiquitin protein family. Our results thus support an overall relation between protein dynamics and conformational changes enabling sequence changes in evolution. More practically, the presented method can be applied to improve protein design predictions by accounting for intrinsic native-state dynamics.     

Molecular mechanisms of activation in CDK2

Cyclin-dependent kinases (CDKs) are enzymes involved in crucial cellular processes. Their biological activity is directly linked to their high conformational variability, which involves large protein conformational rearrangements. We present here the application of an enhancing sampling technique to the study of conformational transitions between the open and closed state of CDKs. The analysis of the conformational intermediates supports the idea that the process is regulated by two important protein regions, which sequentially rearrange in order to allow the protein to reach its final conformation. Furthermore, the two paths involve additional (minor) protein rearrangements which are specific to the paths. Our results show that our procedure can provide reasonable transition pathways between the two protein forms at a very reduced computational cost. The robustness and the simplicity of our approach make it of general application to describe virtually any macromolecular conformational transitions.     

Did the prion protein become vulnerable to misfolding after an evolutionary divide and conquer event?

Despite high sequence identity among mammalian prion proteins (PrPs), mammals have varying rates of susceptibility to prion disease resulting in a so-called species barrier. The species barrier follows no clear pattern, with closely related species or similar sequences being no more likely to infect each other, and remains an unresolved enigma. Variation of the conformationally flexible regions may alter the thermodynamics of the conformational change, commonly referred to as the conformational conversion, which occurs in the pathogenic process of the mammalian prion protein. A conformational ensemble scenario is supported by the species barrier in prion disease and evidence that there are strains of pathogenic prion with different conformations within species. To study how conformational flexibility has evolved in the prion protein, an investigation was undertaken on the evolutionary dynamics of structurally disordered regions in the mammalian prion protein, non-mammalian prion protein that is not vulnerable to prion disease, and remote homologs Doppel and Shadoo. Structural disorder prediction analyzed in an evolutionary context revealed that the occurrence of increased or altered conformational flexibility in mammalian PrPs coincides with key events among PrP, Doppel, and Shadoo. Comparatively rapid evolutionary dynamics of conformational flexibility in the prion protein suggest that the species barrier is not a static phenomenon. A small number of amino acid substitutions can repopulate the conformational ensemble and have a disproportionately large effect on pathogenesis.     

Stretched-Exponential Analysis of Heat-Induced Aggregation of Apo-Concanavalin A

The kinetics of heat-induced aggregation of apo-concanavalin A (aConA) was investigated as a function of temperature and protein concentration by circular dichroism and turbidity. Heat-induced aggregation, as well as conformational change, of aConA was fitted to stretched-exponential equations. The exponent of the conformational change maintained 0.5 despite the protein concentration and temperature, indicating the presence of a common intermediate during the conformational change. After the process, aggregates grew with increasing temperature and initial protein concentration. The reaction order of aggregation was 1.5, indicating that the rate-limiting steps of aConA aggregation involve both conformational change and aggregation.     

A toxic monomeric conformer of the polyglutamine protein

Polyglutamine (polyQ) diseases are classified as conformational neurodegenerative diseases, like Alzheimer and Parkinson diseases, and they are caused by proteins with an abnormally expanded polyQ stretch. However, conformational changes of the expanded polyQ protein and the toxic conformers formed during aggregation have remained poorly understood despite their important role in pathogenesis. Here we show that a beta-sheet conformational transition of the expanded polyQ protein monomer precedes its assembly into beta-sheet-rich amyloid-like fibrils. Microinjection of the various polyQ protein conformers into cultured cells revealed that the soluble beta-sheet monomer causes cytotoxicity. The polyQ-binding peptide QBP1 prevents the toxic beta-sheet conformational transition of the expanded polyQ protein monomer. We conclude that the toxic conformational transition, and not simply the aggregation process itself, is a therapeutic target for polyQ diseases and possibly for conformational diseases in general.     

A model of threshold behavior reveals rescue mechanisms of bystander proteins in conformational diseases

Conformational diseases result from the failure of a specific protein to fold into its correct functional state. The misfolded proteins can lead to the toxic aggregation of proteins. Protein misfolding in conformational diseases often displays a threshold behavior characterized by a sudden shift between nontoxic to toxic levels of misfolded proteins. In some conformational diseases, evidence suggests that misfolded proteins interact with bystander proteins (unfolded and native folded proteins), eliciting a misfolded phenotype. These bystander isomers would follow their normal physiological pathways in absence of misfolded proteins. In this article, we present a general mechanism of bystander and misfolded protein interaction which we have used to investigate how the threshold behavior in protein misfolding is triggered in conformational diseases. Using a continuous flow reactor model of the endoplasmic reticulum, we found that slight changes in the bystander protein residence time in the endoplasmic reticulum or the ratio of basal misfolded to bystander protein inflow rates can trigger the threshold behavior in protein misfolding. Our analysis reveals three mechanisms to rescue bystander proteins in conformational diseases. The results of our model can now help direct experiments to understand the threshold behavior and develop therapeutic strategies targeting the modulation of conformational diseases.     

Reconciling Mediating and Slaving Roles of Water in Protein Conformational Dynamics

Proteins accomplish their physiological functions with remarkably organized dynamic transitions among a hierarchical network of conformational substates. Despite the essential contribution of water molecules in shaping functionally important protein dynamics, their exact role is still controversial. Water molecules were reported either as mediators that facilitate or as masters that slave protein dynamics. Since dynamic behaviour of a given protein is ultimately determined by the underlying energy landscape, we systematically analysed protein self energies and protein-water interaction energies obtained from extensive molecular dynamics simulation trajectories of barstar. We found that protein-water interaction energy plays the dominant role when compared with protein self energy, and these two energy terms on average have negative correlation that increases with increasingly longer time scales ranging from 10 femtoseconds to 100 nanoseconds. Water molecules effectively roughen potential energy surface of proteins in the majority part of observed conformational space and smooth in the remaining part. These findings support a scenario wherein water on average slave protein conformational dynamics but facilitate a fraction of transitions among different conformational substates, and reconcile the controversy on the facilitating and slaving roles of water molecules in protein conformational dynamics.     

Ligand-induced conformational change of a protein reproduced by a linear combination of displacement vectors obtained from normal mode analysis

The conformational change of a protein upon ligand binding was examined by normal mode analysis (NMA) based on an elastic-network model (ENM) for a full-atom system using dihedral angles as independent variables. Specifically, we investigated the extent to which conformational change vectors of atoms from an apo form to a holo form of a protein can be represented by a linear combination of the displacement vectors of atoms in the apo form calculated for the lowest-frequency m normal modes (m=1, 2,…, 20). In this analysis, the latter vectors were best fitted to the former ones by the least-squares method. Twenty-two paired proteins in the holo and apo forms, including three dimer pairs, were examined. The results showed that, in most cases, the conformational change vectors were reproduced well by a linear combination of the displacement vectors of a small number of low-frequency normal modes. The conformational change around an active site was reproduced as well as the entire conformational change, except for some proteins that only undergo significant conformational changes around active sites. The weighting factors for 20 normal modes optimized by the least-squares fitting characterize the conformational changes upon ligand binding for these proteins. The conformational changes sampled around the apo form of a protein by the linear combination of the displacement vectors obtained by ENM-based NMA may help solve the flexible-docking problem of a protein with another molecule because the results presented herein suggest that they have a relatively high probability of being involved in an actual conformational change.     

Conformational changes in the human estrogen receptor observed by (19)F NMR

The (19)F NMR spectra of the 5F-Trp labeled glutathione-S-transferase fusion protein with residues 282-595 of the human estrogen receptor show that there is a distinct conformational change in the protein when estradiol is added to the unliganded protein. Our studies show the empty receptor to have more conformational flexibility than the liganded form. This study shows the applicability of (19)F NMR to study conformational change in large protein systems.     

Relative solvent accessible surface area predicts protein conformational changes upon binding

Protein interactions are often accompanied by significant changes in conformation. We have analyzed the relationships between protein structures and the conformational changes they undergo upon binding. Based upon this, we introduce a simple measure, the relative solvent accessible surface area, which can be used to predict the magnitude of binding-induced conformational changes from the structures of either monomeric proteins or bound subunits. Applying this to a large set of protein complexes suggests that large conformational changes upon binding are common. In addition, we observe considerable enrichment of intrinsically disordered sequences in proteins predicted to undergo large conformational changes. Finally, we demonstrate that the relative solvent accessible surface area of monomeric proteins can be used as a simple proxy for protein flexibility. This reveals a powerful connection between the flexibility of unbound proteins and their binding-induced conformational changes, consistent with the conformational selection model of molecular recognition.     

Selected-fit versus induced-fit protein binding: kinetic differences and mutational analysis

The binding of a ligand molecule to a protein is often accompanied by conformational changes of the protein. A central question is whether the ligand induces the conformational change (induced-fit), or rather selects and stabilizes a complementary conformation from a pre-existing equilibrium of ground and excited states of the protein (selected-fit). We consider here the binding kinetics in a simple four-state model of ligand-protein binding. In this model, the protein has two conformations, which can both bind the ligand. The first conformation is the ground state of the protein when the ligand is off, and the second conformation is the ground state when the ligand is bound. The induced-fit mechanism corresponds to ligand binding in the unbound ground state, and the selected-fit mechanism to ligand binding in the excited state. We find a simple, characteristic difference between the on- and off-rates in the two mechanisms if the conformational relaxation into the ground states is fast. In the case of selected-fit binding, the on-rate depends on the conformational equilibrium constant, whereas the off-rate is independent. In the case of induced-fit binding, in contrast, the off-rate depends on the conformational equilibrium, while the on-rate is independent. Whether a protein binds a ligand via selected-fit or induced-fit thus may be revealed by mutations far from the protein's binding pocket, or other "perturbations" that only affect the conformational equilibrium. In the case of selected-fit, such mutations will only change the on-rate, and in the case of induced-fit, only the off-rate.     

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.     

Membrane protein conformational change dependent on the hydrophobic environment

Two conformational states of the coat protein of the filamentous bacteriophage M13 have been detected in detergent solution by using magnetic resonance techniques. When 3-fluorotyrosine is incorporated in place of the two tyrosine residues in the protein, four 19F nuclear magnetic resonance signals are observed, two for each conformer of the protein. The equilibrium between the two forms can be modulated by pH, temperature, and detergent structure. The rate of interconversion of the isomers is rapid on the minutes time scale but is slow relative to the T1 relaxation time of the fluorine resonances of approximately 50 ms. The conformational change between the conformers results in the perturbation of a basic residue in the protein such that this group has a pKa of approximately 9.5 in one state which shifts to 10.5 or more in the other conformational state. The temperature dependence of the equilibrium suggests an enthalpy difference of about 10 kcal/mol which is offset by entropy to give nearly zero free energy difference between the states at pH 8.3 in deoxycholate solution at room temperature. This suggests a substantial reorganization of the noncovalent interactions defining the two conformational states. The conformational equilibrium is strongly dependent on detergent structure and the presence of phospholipid in the detergent micelle. The results are not consistent with a strong, specific lipid binding to the protein but appear to be consistent with a more general effect of the overall micelle structure on the conformational state of the protein.     

Conformational fluctuations of proteins revealed by variable pressure NMR

With the high-resolution variable-pressure NMR spectroscopy, one can study conformational fluctuations of proteins in a much wider conformational space than hitherto explored by NMR and other spectroscopic techniques. This is because a protein in solution generally exists as a dynamic mixture of conformers mutually differing in partial molar volume, and pressure can select the population of a conformer according to its relative volume. In this review, we describe how variable-pressure NMR can be used to probe conformational fluctuations of proteins in a wide conformational space from the folded to the fully unfolded structures, with actual examples. Furthermore, the newly emerging technique "NMR snapshots" expresses amply fluctuating protein structures as changes in atomic coordinates. Finally, the concept of conformational fluctuation is extended to include intermolecular association leading to amyloidosis.     

Discovering conformational sub-states relevant to protein function

Background

Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these sub-states present significant challenges for their identification and characterization.

Methods and Findings

To overcome these challenges we have developed a new computational technique, quasi-anharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equilibrium simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states associated with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these three proteins, QAA allows identification of conformational sub-states, with critical structural and dynamical features relevant to protein function.

Conclusions

Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function.     

pH-induced conformational transition in the soluble CuA domain of Paracoccus denitrificans cytochrome oxidase

The pH-induced conformational transition in the CuA domain of subunit II of cytochrome oxidase of Paracoccus denitrificans (PdII) has been investigated using various spectroscopic and stopped-flow kinetic methods. UV-visible absorption and circular dichroism studies showed that an increase in pH from 6 to 10 leads to a conformation change with pK(a) = 8.2 associated with the CuA site of the protein. The secondary structure of the protein was, however, shown to remain unchanged in these two conformational states. Thermal and urea-induced unfolding studies showed that the "low-pH" conformation is more stable compared to the "high-pH" conformation of the protein. Moreover, the overall stability of the protein was found to decrease on reduction of the metal centers in the low-pH form, while the oxidation state of the metal centers did not have any significant effect on the overall stability of the protein in the high-pH form. Stopped-flow pH-jump kinetic studies suggested that the conformational transition is associated with a slow deprotonation step followed by fast conformational equilibrium. The results are discussed in the light of understanding the pH-induced conformational change in the beta-barrel structure of the protein and its effect on the coordination geometry of the metal site.     

Osmolytes modulate conformational exchange in solvent‐exposed regions of membrane proteins

Site‐directed spin labeling (SDSL) was used to investigate local structure and conformational exchange in two bacterial outer‐membrane TonB‐dependent transporters, BtuB and FecA. Protecting osmolytes, such as polyethylene glycols (PEGs) are known to modulate a substrate‐dependent conformational equilibrium in the energy coupling motif (Ton box) of BtuB. Here, we demonstrate that a segment that is N‐terminal to the Ton box in BtuB, is in conformational exchange between ordered and disordered states with or without substrate. Protecting osmolytes shift this equilibrium to favor the more ordered, folded state. However, a segment of BtuB that is C‐terminal to the Ton box that is not solvent exposed is insensitive to PEGs. Protecting osmolytes also modulate a conformational equilibrium in the Ton box of FecA, with larger molecular weight PEGs producing the largest shifts in the conformational free energy. These data indicate that solvent‐exposed regions of these transporters undergo conformational exchange and that regions of these transporters that are involved in protein–protein interactions sample multiple conformational substates. The sensitivity to solute provides an explanation for differences seen between two high‐resolution structures of BtuB, which each likely represent one conformation from a subset of states that are normally sampled by the protein. This work also illustrates how SDSL and osmolytes may be used to characterize and quantitate conformational equilibria in membrane proteins.     

Driving Protein Conformational Cycles in Physiology and Disease: “Frustrated” Amino Acid Interaction Networks Define Dynamic Energy Landscapes

A general framework by which dynamic interactions within a protein will promote the necessary series of structural changes, or “conformational cycle,” required for function is proposed. It is suggested that the free-energy landscape of a protein is biased toward this conformational cycle. Fluctuations into higher energy, although thermally accessible, conformations drive the conformational cycle forward. The amino acid interaction network is defined as those intraprotein interactions that contribute most to the free-energy landscape. Some network connections are consistent in every structural state, while others periodically change their interaction strength according to the conformational cycle. It is reviewed here that structural transitions change these periodic network connections, which then predisposes the protein toward the next set of network changes, and hence the next structural change. These concepts are illustrated by recent work on tryptophan synthase. Disruption of these dynamic connections may lead to aberrant protein function and disease states.