Calorimetric evidence for a two-state unfolding of the beta-hairpin peptide trpzip4

beta-Sheets are a common secondary structural element found in proteins. The difficulty in studying beta-sheet folding and stability is that their formation is often dependent on the tertiary structural environment within the protein. However, the discovery of water-soluble beta-hairpin peptides has allowed them to be used as model systems because they represent the smallest units of beta-sheet structure independent of tertiary structural context. Trpzip4 has been used as a model beta-hairpin peptide to study beta-hairpin folding and stability because it is highly soluble in aqueous solutions, maintains its monomeric state, and shows reversible cooperative thermal unfolding. The previously determined thermodynamic parameters for trpzip4 thermal unfolding vary depending on the spectroscopic probe used, which questions the assumption that trpzip4 unfolds in a two-state manner. Here we provide direct calorimetric evidence that the unfolding of trpzip4 follows a two-state unfolding mode. Furthermore, the thermal unfolding of trpzip4 monitored using near- and far-UV-CD yielded thermodynamic parameters similar to those determined calorimetrically, providing additional evidence for a two-state unfolding mode.     

Computer simulations of the refolding of sperm whale apomyoglobin from high-temperature denaturated state

The refolding mechanism of apomyoglobin (apoMb) subsequent to high-temperature unfolding has been examined using computer simulations with atomic level detail. The folding of this protein has been extensively studied experimentally, providing a large database of folding parameters which can be probed using simulations. In the present study, 4-folding trajectories of apoMb were computed starting from coiled structures. A crystal structure of sperm whale myoglobin taken from the Protein Data Bank was used to construct the final native conformation by removal of the heme group followed by energy optimization. The initial unfolded conformations were obtained from high-temperature molecular dynamics simulations. Room-temperature refolding trajectories at neutral pH were obtained using the stochastic difference equation in length algorithm. The folding trajectories were compared with experimental results and two previous molecular dynamics studies at low pH. In contrast to the previous simulations, an extended intermediate with large helical content was not observed. In the present study, a structural collapse occurs without formation of helices or native contacts. Once the protein structure is more compact (radius of gyration<18 A) secondary and tertiary structures appear. These results suggest that apoMb follows a different folding pathway after high-temperature denaturation.     

Denatured-state ensemble and the early-stage folding of the G29A mutant of the B-domain of protein A

The folding mechanism of the G29A mutant of the B-domain of protein A (BdpA) has been studied by all-atom molecular dynamics simulation using AMBER force field (ff03) and generalized Born continuum solvent model. Started from the extended chain conformation, a total of 16 simulations (400 ns each) at 300 K captured some early folding events of the G29A mutant of BdpA. In one of the 16 trajectories, the G29A mutant folded within 2.8 A (root mean square) of the wild-type NMR structure. We observed that the fast burial of hydrophobic residues was the driving force to bring the distant residues into close proximity. The initiation of the helix I and III occurred during the stage of hydrophobic collapse. The initiation and growth of the helix II was slow. Both the secondary structure formation and the development of the native tertiary contacts suggested a multistage folding process. Clustering analysis indicated that two helix species (helices I and III) could be intermediates. Further analysis revealed that the hydrophobic residues of partially folded helix II formed nativelike hydrophobic contacts with helices I and III that stabilized a nativelike state and delayed the completion of folding of the entire protein. The details of the early folding process were compared with other theoretical and experimental studies. It was found that a nativelike hydrophobic cluster was formed by residues including F(30), I(31), L(34), L(44), L(45), and A(48) that prevented further development of the native structures, and breaking the hydrophobic cluster like this one contributed to the rate-limiting step. This was in complete agreement with the recent kinetic measurements in which mutations of these residues to Gly and Ala substantially increased the folding rates by as much as 60 times. Apparently, destabilization of nonnative states dramatically enhanced the folding rates.     

Atomistic mechanism of protein denaturation by urea

Effects of urea on protein stability have been studied from all-atom molecular dynamics simulations of ubiquitin, G311 protein, and immunoglobulin binding domain (B1) of streptococcal protein G (GB1) in water and 8 M aqueous urea solution. The mechanism of the change in the solvent environment and the early events in protein unfolding by urea have been identified with emphasis on the change in the interactions of hydrophilic and hydrophobic parts of the protein by calculating the potential of mean force (PMF). Urea replaces the protein-protein and protein-water contacts by forming stronger contacts with the protein, which is indicated by the longer survival times of the protein-urea hydrogen bonds.     

Folding transition-state and denatured-state ensembles of FSD-1 from folding and unfolding simulations

Characterization of the folding transition-state ensemble and the denatured-state ensemble is an important step toward a full elucidation of protein folding mechanisms. We report herein an investigation of the free-energy landscape of FSD-1 protein by a total of four sets of folding and unfolding molecular dynamics simulations with explicit solvent. The transition-state ensemble was initially identified from unfolding simulations at 500 K and was verified by simulations at 300 K starting from the ensemble structures. The denatured-state ensemble and the early-stage folding were studied by a combination of unfolding simulations at 500 K and folding simulations at 300 K starting from the extended conformation. A common feature of the transition-state ensemble was the substantial formation of the native secondary structures, including both the alpha-helix and beta-sheet, with partial exposure of the hydrophobic core in the solvent. Both the native and non-native secondary structures were observed in the denatured-state ensemble and early-stage folding, consistent with the smooth experimental melting curve. Interestingly, the contact orders of the transition-state ensemble structures were similar to that of the native structure and were notably lower than those of the compact structures found in early-stage folding, implying that chain and topological entropy might play significant roles in protein folding. Implications for FSD-1 folding mechanisms and the rate-limiting step are discussed. Analyses further revealed interesting non-native interactions in the denatured-state ensemble and early-stage folding and the possibility that destabilization of these interactions could help to enhance the stability and folding rate of the protein.     

Single Molecule Force Spectroscopy Reveals Critical Roles of Hydrophobic Core Packing in Determining the Mechanical Stability of Protein GB1

Understanding molecular determinants of protein mechanical stability is important not only for elucidating how elastomeric proteins are designed and functioning in biological systems but also for designing protein building blocks with defined nanomechanical properties for constructing novel biomaterials. GB1 is a small α/β protein and exhibits significant mechanical stability. It is thought that the shear topology of GB1 plays an important role in determining its mechanical stability. Here, we combine single molecule atomic force microscopy and protein engineering techniques to investigate the effect of side chain reduction and hydrophobic core packing on the mechanical stability of GB1. We engineered seven point mutants and carried out mechanical ?-value analysis of the mechanical unfolding of GB1. We found that three mutations, which are across the surfaces of two subdomains that are to be sheared by the applied stretching force, in the hydrophobic core (F30L, Y45L, and F52L) result in significant decrease in mechanical unfolding force of GB1. The mechanical unfolding force of these mutants drop by 50-90 pN compared with wild-type GB1, which unfolds at around 180 pN at a pulling speed of 400 nm/s. These results indicate that hydrophobic core packing plays an important role in determining the mechanical stability of GB1 and suggest that optimizing hydrophobic interactions across the surfaces that are to be sheared will likely be an efficient method to enhance the mechanical stability of GB1 and GB1 homologues.     

Evidence of a two-stage thermal denaturation process in lysozyme: a Raman scattering and differential scanning calorimetry investigation

Raman spectroscopy (in the low-frequency range and the amide I band region) and modulated differential scanning calorimetry investigations have been used to analyze temperature-induced structural changes in lysozyme dissolved in 1H2O and 2H2O in the thermal denaturation process. Low-frequency Raman data reveal a change in tertiary structure without concomitant unfolding of the secondary structure. Calorimetric data show that this structural change is responsible for the configurational entropy change associated with the strong-to-fragile liquid transition and correspond to about 1/3 of the native-denaturated transition enthalpy. This is the first stage of the thermal denaturation which is a precursor of the secondary structure change and is determined to be strongly dependent on the stability of the hydrogen-bond network in water. Low-frequency Raman spectroscopy provides information on the flexibility of the tertiary structure (in the native state and the transient folding state) in relation to the fragility of the mixture. The unfolding of the secondary structure appears as a consequence of the change in the tertiary structure and independent of the solvent. Protein conformational stability is directly dependent on the stability of the native tertiary structure. The structural transformation of tertiary structure can be detected through the enhanced 1H/2H exchange inhibited in native proteins. Taking into account similar features reported in the literature observed for different proteins it can be considered that the two-stage transformation observed in lysozyme dissolved in water is a general mechanism for the thermal denaturation of proteins.     

The role of plastic beta-hairpin and weak hydrophobic core in the stability and unfolding of a full sequence design protein

In this study, the thermal stability of a designed alpha/beta protein FSD (full sequence design) was studied by explicit solvent simulations at three moderate temperatures, 273 K, 300 K, and 330 K. The average properties of the ten trajectories at each temperature were analyzed. The thermal unfolding, as judged by backbone root-mean-square deviation and percentage of native contacts, was displayed with increased sampling outside of the native basin as the temperature was raised. The positional fluctuation of the hairpin residues was significantly higher than that of the helix residues at all three temperatures. The hairpin segment displayed certain plasticity even at 273 K. Apart from the terminal residues, the highest fluctuation was shown in the turn residues 7-9. Secondary structure analysis manifested the structural heterogeneity of the hairpin segment. It was also revealed by the simulation that the hydrophobic core was vulnerable to thermal denaturation. Consistent with the experiment, the I7Y mutation in the double mutant FSD-EY (FSD with mutations Q1E and I7Y) dramatically increased the protein stability in the simulation, suggesting that the plasticity of the hairpin can be partially compensated by a stronger hydrophobic core. As for the unfolding pathway, the breathing of the hydrophobic core and the separation of the two secondary structure elements (alpha helix and beta hairpin) was the initiation step of the unfolding. The loss of global contacts from the separation further destabilized the hairpin structure and also led to the unwinding of the helix.     

Performance comparison of generalized born and Poisson methods in the calculation of electrostatic solvation energies for protein structures

This study compares generalized Born (GB) and Poisson (PB) methods for calculating electrostatic solvation energies of proteins. A large set of GB and PB implementations from our own laboratories as well as others is applied to a series of protein structure test sets for evaluating the performance of these methods. The test sets cover a significant range of native protein structures of varying size, fold topology, and amino acid composition as well as nonnative extended and misfolded structures that may be found during structure prediction and folding/unfolding studies. We find that the methods tested here span a wide range from highly accurate and computationally demanding PB-based methods to somewhat less accurate but more affordable GB-based approaches and a few fast, approximate PB solvers. Compared with PB solvation energies, the latest, most accurate GB implementations were found to achieve errors of 1% for relative solvation energies between different proteins and 0.4% between different conformations of the same protein. This compares to accurate PB solvers that produce results with deviations of less than 0.25% between each other for both native and nonnative structures. The performance of the best GB methods is discussed in more detail for the application for force field-based minimizations or molecular dynamics simulations.     

Evidence of an intermediate and parallel pathways in protein unfolding from single-molecule fluorescence

Determining how proteins fold into their native structures is a subject of great importance, since ultimately it will allow protein structure and function to be predicted from primary sequence data. In addition, there is now a clear link between protein unfolding and misfolding events and many disease states. However, since proteins fold over rugged, multidimensional energy landscapes, this is a challenging experimental and theoretical problem. Single-molecule fluorescence methods developed over the past decade have the potential to follow the unfolding/folding of individual molecules. Mapping out the landscape without ensemble averaging will enable the identification of intermediate states which may not be significantly populated, in addition to the presence of multiple pathways. To date, there have been only a limited number of single-molecule folding/unfolding studies under nonequilibrium conditions and no intermediates have been observed. Here, for the first time, we present a single-molecule study of the unfolding of a large autofluorescent protein, Citrine, a variant of green fluorescent protein. Single-molecule fluorescence techniques are used to directly detect an intermediate on the unfolding/folding pathway and the existence of parallel unfolding pathways. This work, and the novel methods used, shows that single-molecule fluorescence can now provide new, hitherto experimentally inaccessible, insights into the folding/unfolding of proteins.     

Thermal stability and folding kinetics analysis of disordered protein,securin

Lacking a stable tertiary structure, intrinsically disordered proteins (IDPs) possess particular functions in cell regulation, signaling, and controlling pathways. The study of their unique structural features, thermal stabilities, and folding kinetics is intriguing. In this study, an identified IDP, securin, was used as a model protein. By using a quasi-static five-step (on-path) folding process, the function of securin was restored and analyzed by isothermal titration calorimetry. Fluorescence spectroscopy and particle size analysis indicated that securin possessed a compact hydrophobic core and particle size. The glass transition of securin was characterized using differential scanning microcalorimetry. Furthermore, the folding/unfolding rates (k_obs) of securin were undetectable, implying that the folding/unfolding rate is very fast and that the conformation of securin is sensitive to solvent environmental change. Therefore, securin may fold properly under specific physiological conditions. In summary, the thermal glass transition behavior and undetectable k_obs of folding/unfolding reactions may be two of the indices of IDP.     

Crystallographic B-factors highlight energetic frustration in aldolase folding

Kinetic simulations of the folding and unfolding of the mammalian TIM barrel protein aldolase were conducted to determine if a minimalist monomeric Gō model, using the native structure to determine attractive energies in the protein chain, could capture the experimentally determined folding pathway. The folding order, that is, the order in which different secondary structures fold, between the Gō model simulations and that from hydrogen-deuterium exchange experiments, did not agree. To explain this discrepancy, two alternate variant of the basic Gō model were simulated: (1) a monomer Gō model with native contact energies weighted by a statistical potential (SP model) and (2) a monomer Gō model with native contact energies inversely weighted by crystallographic B factors (B model). The B model demonstrated the best agreement between simulation and experiments. The success of the B model is attributed to the ability of B factors to highlight local energetic frustration in the aldolase structure which results in weaker native contacts in these frustrated regions. The predictive success of the B model also reveals the potential use of B factor information for energetic weighting in general protein modeling.     

Transient tertiary structure formation within the ribosome exit port

The exit tunnel of the ribosome is commonly considered to be sufficiently narrow that co-translational folding can begin only when specific segments of nascent chains are fully extruded from the tunnel. Here we show, on the basis of molecular simulations and comparison with experiment, that the long-range contacts essential for initiating protein folding can form within a nascent chain when it reaches the last 20 ? of the exit tunnel. We further show that, in this "exit port", a significant proportion of native and non-native tertiary structure can form without steric overlap with the ribosome itself, and provide a library of structural elements that our simulations predict can form in the exit tunnel and is amenable to experimental testing. Our results show that these elements of folded tertiary structure form only transiently and are at their midpoints of stability at the boundary region between the inside and the outside of the tunnel. These findings provide a framework for interpreting a range of recent experimental studies of ribosome nascent chain complexes and for understanding key aspects of the nature of co-translational folding.     

Topology-based models and NMR structures in protein folding simulations

Topology-based interaction potentials are simplified models that use the native contacts in the folded structure of a protein to define an energetically unfrustrated folding funnel. They have been widely used to analyze the folding transition and pathways of different proteins through computer simulations. Obviously, they need a reliable, experimentally determined folded structure to define the model interactions. In structures elucidated through NMR spectroscopy, a complex treatment of the raw experimental data usually provides a series of models, a set of different conformations compatible with the available experimental data. Here, we use an efficient coarse-grained simulation technique to independently consider the contact maps from every different NMR model in a protein whose structure has been resolved by the use of NMR spectroscopy. For lambda-Cro repressor, a homodimeric protein, we have analyzed its folding characteristics with a topology-based model. We have focused on the competition between the folding of the individual chains and their binding to form the final quaternary structure. From 20 different NMR models, we find a predominant three-state folding behavior, in agreement with experimental data on the folding pathway for this protein. Individual NMR models, however, show distinct characteristics, which are analyzed both at the level of the interplay between tertiary/quaternary structure formation and also regarding the thermal stability of the tertiary structure of every individual chain.     

How Photoisomerization Drives Peptide Folding and Unfolding: Insights from QM/MM and MM Dynamics Simulations

Photoswitchable azobenzene cross‐linkers can control the folding and unfolding of peptides by photoisomerization and can thus regulate peptide affinities and enzyme activities. Using quantum mechanics/molecular mechanics (QM/MM) methods and classical MM force fields, we report the first molecular dynamics simulations of the photoinduced folding and unfolding processes in the azobenzene cross‐linked FK‐11 peptide. We find that the interactions between the peptide and the azobenzene cross‐linker are crucial for controlling the evolution of the secondary structure of the peptide and responsible for accelerating the folding and unfolding events. They also modify the photoisomerization mechanism of the azobenzene cross‐linker compared with the situation in vacuo or in solution.     

Atomistic analysis of pseudoknotted RNA unfolding

As important functional structures, RNA pseudoknots provide excellent models for studying the interplay between secondary and tertiary structures and the roles of triplexes, noncanonical interactions, and coaxial stacking in the folding/unfolding process. Here we report a first atomistic and statistical analysis of the unfolding of the pseudoknot within gene 32 mRNA of bacteriophage T2. Multiple unfolding pathways, diverse transition states, and various intermediate structures were observed. Water molecules were found to be coupled with the unfolding process via the expulsion or concurrent mechanism.     

Influence of temperature, friction, and random forces on folding of the B-domain of staphylococcal protein A: all-atom molecular dynamics in implicit solvent

The influences of temperature, friction, and random forces on the folding of protein A have been analyzed. A series of all-atom molecular dynamics folding simulations with the Amber ff99 potential and Generalized Born solvation, starting from the fully extended chain, were carried out for temperatures from 300 to 500 K, using (a) the Berendsen thermostat (with no explicit friction or random forces) and (b) Langevin dynamics (with friction and stochastic forces explicitly present in the system). The simulation temperature influences the relative time scale of the major events on the folding pathways of protein A. At lower temperatures, helix 2 folds significantly later than helices 1 and 3. However, with increasing temperature, the folding time of helix 2 approaches the folding times of helices 1 and 3. At lower temperatures, the complete formation of secondary and tertiary structure is significantly separated in time whereas, at higher temperatures, they occur simultaneously. These results suggest that some earlier experimental and theoretical observations of folding events, e.g., the order of helix formation, could depend on the temperature used in those studies. Therefore, the differences in temperature used could be one of the reasons for the discrepancies among published experimental and computational studies of the folding of protein A. Friction and random forces do not change the folding pathway that was observed in the simulations with the Berendsen thermostat, but their explicit presence in the system extends the folding time of protein A.