Folding Studies Research Articles

The B domain of protein A retains residual structures in 6 M guanidinium chloride as revealed by hydrogen/deuterium-exchange NMR spectroscopy.

The characterization of residual structures persistent in unfolded proteins is an important issue in studies of protein folding, because the residual structures present, if any, may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the residual structures of the isolated B domain (BDPA) of staphylococcal protein A in 6M guanidinium chloride. BDPA is a small three-helix-bundle protein, and until recently its folding/unfolding reaction has been treated as a simple two-state process between the native and the fully unfolded states. We employed a dimethylsulfoxide (DMSO)-quenched hydrogen/deuterium (H/D)-exchange 2D NMR techniques with the use of spin desalting columns, which allowed us to investigate the H/D-exchange behavior of individually identified peptide amide (NH) protons. We obtained H/D-exchange protection factors of the 21 NH protons that form an α-helical hydrogen bond in the native structure, and the majority of these NH protons were significantly protected with a protection factor of 2.0-5.2 in 6M guanidinium chloride, strongly suggesting that these weakly protected NH protons form much stronger hydrogen bonds under native folding conditions. The results can be used to deduce the structure of an early folding intermediate, when such an intermediate is shown by other methods. Among three native helical regions, the third helix in the C-terminal side was highly protected and stabilized by side-chain salt bridges, probably acting as the folding initiation site of BDPA. The present results are discussed in relation to previous experimental and computational findings on the folding mechanisms of BDPA.

Applications of hydrogen deuterium exchange ensemble reweighting to model native protein ensembles, macromolecular interactions and protein drug interactions.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful approach to probe the structural dynamics of a protein in solution. It simultaneously provides time-resolved information on the structural environment of the amide hydrogen of every residue of a protein except Prolines. As such, HDX-MS has been successfully applied to provide insight into the characterization of native state ensembles of proteins, macromolecular interactions, protein-small molecule interactions or protein folding and unfolding studies. However, it remains largely limited to peptide-level resolution information. On the other hand, molecular dynamics (MD) simulations provide a high-resolution view of a protein's structural dynamics by simulating the motion of a protein's atoms over time starting from a high-resolution structure. However, the time scales accessible to MD simulations are limited and can lead to inadequate exploration of a protein's conformational landscape. To bridge this gap, efforts have aimed at developing quantitative and integrative approaches that leverage HDX-MS data to assess and refine computationally generated structural ensembles. HDX ensemble reweighting (HDXer) is one such approach which uses a post hoc maximum entropy approach to adjusts the weights of individual frames of a structural ensemble to conform to HDX-MS data. We had previously applied HDXer to model the native ensemble of the cytoplasmic heme binding protein, PhuS. Here, we have validated the proposed model ensemble by using structure-based drug design approach to successfully target novel, cryptic drug binding sites that would have otherwise not been targetable using HDX-MS or MD simulations alone. Finally, we demonstrate the broader applicability of such HDX-MS guided integrative approach to model macromolecular interactions, missing electron density and protein-small molecule drug interactions

Creating Selenocysteine-Specific Reporters Using Inteins.

The selenium moiety in selenocysteine (Sec) imparts enhanced chemical properties to this amino acid and ultimately the protein in which it is inserted. These characteristics are attractive for designing highly active enzymes or extremely stable proteins and studying protein folding or electron transfer, to name a few. There are also 25 human selenoproteins, of which many are essential for our survival. The ability to create or study these selenoproteins is significantly hindered by the inability to easily produce them. Engineering translation has yielded simpler systemsto facilitate site-specific insertion of Sec; however, Ser misincorporation remains problematic. Therefore, we have designed two Sec-specific reporters which promote high-throughput screening of Sec translation systems to overcome this barrier. This protocol outlines the workflow to engineer these Sec-specific reporters, with the application to any gene of interest and the ability to transfer this strategy to any organism.

Metal-binding and circular permutation-dependent thermodynamic and kinetic stability of azurin.

Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key β-barrel, β-sandwich, β-α-β, and all α-Greek key. CP mainly affects the protein stability and unfolding kinetics, while folding kinetics remains mostly unaltered. However, the effect of CP on metalloproteins is yet to be elaborately studied. The active site of metalloproteins poses an additional complexity in studying protein folding. Here, we investigate a CP variant (cpN42) of azurin-in both metal-free and metal-bound (holo) forms. As observed earlier in other proteins, apo-forms of wild-type (WT) and cpN42 fold with similar rates. In contrast, zinc-binding accelerates the folding of WT but decelerates the folding of cpN42. On zinc-binding, the spontaneous folding rate of WT increases by >250 times that of cpN42, which is unprecedented and the highest for any CP to date. On the other hand, zinc-binding reduces the spontaneous unfolding rate of cpN42 by ~100 times, making the WT and CP azurins unfold at similar rates. Our study demonstrates metal binding as a novel way to modulate the unfolding and folding rates of CPs compared to their WT counterparts. We hope our study increases the understanding of the effect of CP on the folding mechanism and energy landscape of metalloproteins.

Differentiating between the sequence of structural events on alternative pathways of folding of a heterodimeric protein.

Distinguishing between competing pathways of folding of a protein, on the basis of how they differ in their progress of structure acquisition, remains an important challenge in protein folding studies. A previous study had shown that the heterodimeric protein, double chain monellin (dcMN) switches between alternative folding pathways upon a change in guanidine hydrochloride (GdnHCl) concentration. In the current study, the folding of dcMN has been characterized by the pulsed hydrogen exchange (HX) labeling methodology used in conjunction with mass spectrometry. Quantification of the extent to which folding intermediates accumulate and then disappear with time of folding at both low and high GdnHCl concentrations, where the folding pathways are known to be different, shows that the folding mechanism is describable by a triangular three-state mechanism. Structural characterization of the productive folding intermediates populated on the alternative pathways has enabled the pathways to be differentiated on the basis of the progress of structure acquisition that occurs on them. The intermediates on the two pathways differ in the extent to which the α-helix and the rest of the β-sheet have acquired structure that is protective against HX. The major difference is, however, that β2 has not acquired any protective structure in the intermediate formed on one pathway, but it has acquired significant protective structure in the intermediate formed on the alternative pathway. Hence, the sequence of structural events is different on the two alternative pathways.

Scanning pH-metry for Observing Reversibility in Protein Folding.

One of the main factors affecting protein structure in solution is pH. Traditionally, to study pH-dependent conformational changes in proteins, the concentration of the H+ ions is adjusted manually, complicating real-time analyses, hampering dynamic pH regulation, and consequently leading to a limited number of tested pH levels. Here, we present a programmable device, a scanning pH-meter, that can automatically generate different types of pH ramps and waveforms in a solution. A feedback loop algorithm calculates the required flow rates of the acid/base titrants, allowing one, for example, to generate periodic pH sine waveforms to study the reversibility of protein folding by fluorescence spectroscopy. Interestingly, for some proteins, the fluorescence intensity profiles recorded in such a periodically oscillating pH environment display hysteretic behavior indicating an asymmetry in the sequence of the protein unfolding/refolding events, which can most likely be attributed to their distinct kinetics. Another useful application of the scanning pH-meter concerns coupling it with an electrospray ionization mass spectrometer to observe pH-induced structural changes in proteins as revealed by their varying charge-state distributions. We anticipate a broad range of applications of the scanning pH-meter developed here, including protein folding studies, determination of the optimum pH for achieving maximum fluorescence intensity, and characterization of fluorescent dyes and other synthetic materials.

Proteins from Thermophilic Thermus thermophilus Often Do Not Fold Correctly in a Mesophilic Expression System Such as Escherichia coli.

Majority of protein structure studies use Escherichia coli (E. coli) and other model organisms as expression systems for other species’ genes. However, protein folding depends on cellular environment factors, such as chaperone proteins, cytoplasmic pH, temperature, and ionic concentrations. Because of differences in these factors, especially temperature and chaperones, native proteins in organisms such as extremophiles may fold improperly when they are expressed in mesophilic model organisms. Here we present a methodology of assessing the effects of using E. coli as the expression system on protein structures. We compare these effects between eight mesophilic bacteria and Thermus thermophilus (T. thermophilus), a thermophile, and found that differences are significantly larger for T. thermophilus. More specifically, helical secondary structures in T. thermophilus proteins are often replaced by coil structures in E. coli. Our results show unique directionality in misfolding when proteins in thermophiles are expressed in mesophiles. This indicates that extremophiles, such as thermophiles, require unique protein expression systems in protein folding studies.

Umbrella sampling with machine learning potentials applied for solid phase transition of GeSbTe

The free energy difference is the key role to predict reaction rates of solid phase transition. Umbrella sampling is an effective method for enhanced sampling, especially in the region with high potential energy, and has been widely applied in studies of protein folding and aggregation, etc. Here, we demonstrated a strategy that integrates umbrella sampling with machine learning potentials (US–MLPs) for the study of solid phase transition. The method succeeded in theoretically investigating the phase transition process of GeSbTe from vacancy ordered cubic phase to quasi–amorphous phase. The initial and final geometries of the phase transition process were obtained by using ab initio molecular dynamics (AIMD). Subsequently, the free energy barrier and location of transition state of the above phase transition process were investigated by the US–MLPs method. During the sampling process, the constrained molecular dynamics (MD) combining with MLPs lasted 51 ns and generated 510,000 structure. For comparing with experiment, our method shows advantages over previous simulation method such as the climbing image nudged elastic band (CI–NEB) method.

Abstract 3988: Identification of HER4 mutations with potential as biomarkers of response to neratinib

Abstract Background: HER4 is a member of the human epidermal growth factor receptor (HER/ErbB) family of receptor tyrosine kinases. HER4 is activated through ligand binding, transmitting intracellular signals by homodimerizing, or by heterodimerizing with other family members (EGFR, HER2, HER3). The role of HER4 in cancer or normal tissue has not been studied as comprehensively as EGFR and HER2, potentially due to a more complex biology that includes four different isoforms and different stability/tissue-specific expression patterns, and oncogenic or oncosuppressor roles. The oral, irreversible pan-HER (EGFR, HER2, HER4) tyrosine kinase inhibitor (TKI) neratinib (NER) is clinically active against HER2-mutant and HER2-amplified cancers. Using publicly available datasets including cBioPortal and COSMIC and predicted structural and functional impacts, this study aimed to identify HER4 mutations with the greatest oncogenic potential that may have value as potential biomarkers of response to NER. Methods: The frequency of HER4 mutations across 33 cancer types was determined by using the clinical database cBioPortal and TCGA PanCancer Atlas datasets (n=10967). A shortlist of potentially oncogenic HER4 mutations was created based on the occurrence in cBioPortal and COSMIC databases, annotation as putative driver and cancer hotspot, structural and functional impacts according to OncoKB, Mutation Assessor, SIFT and Polyphen-2 software, and HER4 protein folding studies. Analysis was carried out using the R-studio statistical environment. Results: HER4 amplification is rare in cancer, with the majority of genetic changes reported as point mutations; particularly missense (31.93%), synonymous (6.43%) and nonsense (3%) substitutions. Melanoma and esophagogastric (EG) cancers showed the highest HER4 alteration frequency, 17.34% and 13.42%, respectively, across different cancer types in the cBioPortal database. Melanoma and EG cancers showed the second and third highest absolute count of HER4 genomic alterations (n=77 and n=69, respectively). Based on the criteria outlined in methods, a priority list of HER4 mutations with greatest oncogenic potential was curated for melanoma (HER4 R106C, R306C, R711C, G936E, F1003I), stomach (HER4 C217R, S774N, K1002R, Y1150C) and esophageal (HER4 R488W, S449Y) adenocarcinomas. Mutations at these sites, located across ligand-binding, dimerization and intracellular signaling domains, have the potential to impact HER4 activity and thus possibly act as biomarkers of response to an irreversible pan-HER TKI like NER. Conclusions: These data suggest that melanoma and EG cancers are the cancer types with the highest rate of HER4 mutations. In vitro functional characterization of the identified HER4 mutations is now required to further explore their value as biomarkers of response to NER. Citation Format: Marta Valenti, Neil T. Conlon, Lisa D. Eli, Irmina Diala, John Crown, Denis M. Collins. Identification of HER4 mutations with potential as biomarkers of response to neratinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3988.

Pyrene Excimer Formation (PEF) and Its Application to the Study of Polypeptide Dynamics.

This Perspective describes how the fluorescence blob model (FBM) has been developed and applied over the past 30 years to characterize the long-range backbone dynamics (LRBD) of polymers in solution. In these experiments, the polymers are randomly labeled with the dye pyrene, which forms an excimer upon the encounter between an excited and a ground-state pyrenyl label inside a finite subvolume of the polymer coil referred to as a blob representing the volume probed by the excited pyrene. By compartmentalizing the polymer coil into a cluster of identical blobs, FBM analysis of the fluorescence decays acquired with the polymers yields the number Nblob of structural units inside a blob. Since a flexible or rigid backbone will result in an Nblob that is either large or small, Nblob can be used as a measure of the flexibility of a given polymer. After having established that these experiments based on pyrene excimer formation (PEF) yielded quantitative information about the LRBD of a variety of polymers in solution, control experiments were carried out to characterize the effects that different molecular variables, such as the side-chain size (SCS) of a structural unit or the length of the linker connecting pyrene to the polymeric backbone, had on the parameters retrieved with the FBM. At this point, the FBM was applied to study the LRBD of polypeptides prepared from racemic mixtures of amino acids (aa's). These studies led to the establishment of simple rules that could be developed into mathematical equations to describe the LRBD of polypeptides. The Nblob values retrieved from the FBM analysis of the fluorescence decays acquired with the pyrene-labeled polypeptides could then be employed to predict the total conformational search time (τtcs) of any polypeptide based on their sequence. Strong correlations were found between the predicted τtcs and the experimental folding times of 145 proteins. The good quality of these correlations suggests that the blob-based approach described in this report might represent an interesting mathematical means for studying protein folding.

Reconstruction of the Free Energy Profile for SUMO1 from Nonequilibrium Single-Molecule Pulling Experiments

Free energy profiles form the cornerstone in the study of protein folding and function. In this study, the free energy profile of SUMO1 protein is directly reconstructed using an extension of the Jarzynski equality from atomic force microscope (AFM) based single-molecule force spectroscopy (SMFS) experiments. SUMO1 is a ubiquitin-like posttranslational modifier protein having a β clamp motif in its structure, imparting it with mechanical stability. We use the Jarzynski equality to obtain the equilibrium free energy profile from repeated nonequilibrium single-molecule pulling experiments. Indeed, the free energy values determined by the Jarzynski equality are lesser than the normal work average at all extensions. The free energy profiles constructed for the two velocities (100 and 400 nm/s) overlap with each other. The unfolding free energy barrier is estimated to be ∼7.5 kcal/mol. We anticipate that the Jarzynski equality can be applied in a similar manner to other ubiquitin-like proteins to extract their differences in the free energy profile, and hence, the effect of sequence diversity of structurally homologous proteins on the free energy landscape can be studied.

Annealing synchronizes the 70S ribosome into a minimum-energy conformation

Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70S ribosome from Escherichia coli as a model, wherein the 30S subunit undergoes a thermally driven intersubunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70S ribosome homogeneity and improves local resolution on the 30S subunit. After annealing, the 70S ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes. Manifold-based analysis further indicates that the annealed 70S ribosome takes a narrow conformational distribution and exhibits a minimum-energy state in the free-energy landscape. Our experimental results offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing shows great potential for synchronizing proteins on a single-molecule level and can be extended to study protein folding and explore conformational and energy landscapes.

Residue-Frustration-Based Prediction of Protein-Protein Interactions Using Machine Learning.

The study of protein-protein interactions (PPIs) is important in understanding the function of proteins. However, it is still a challenge to investigate the transient protein-protein interaction by experiments. Hence, the computational prediction for protein-protein interactions draws growing attention. Statistics-based features have been widely used in the studies of protein structure prediction and protein folding. Due to the scarcity of experimental data of PPI, it is difficult to construct a conventional statistical feature for PPI prediction, and the application of statistics-based features is very limited in this field. In this paper, we explored the application of frustration, a statistical potential, in PPI prediction. By comparing the energetic contribution of the extra stabilization energy from a given residue pair in the native protein with the statistics of the energies, we obtained the residue pair's frustration index. By calculating the number of residue pairs with a high frustration index, the highly frustrated density, a residue-frustration-based feature, was then obtained to describe the tendency of residues to be involved in PPI. Highly frustrated density, as well as structure-based features, were then used to describe protein residues and combined with the long short-term memory (LSTM) neural network to predict PPI residue pairs. Our model correctly predicted 75% dimers when only the top 2‰ residue pairs were selected in each dimer. Our model, which considers the statistics-based features, is significantly different from the models based on the chemical features of residues. We found that frustration can effectively describe the tendency of residue to be involved in PPI. Frustration-based features can replace chemical features to combine with machine learning and realize the better performance of PPI prediction. It reveals the great potential of statistical potential such as frustration in PPI prediction.

The ClpX translocase and its application to the study of vectorial protein folding in vitro

In vitro protein refolding upon dilution from a chemical denaturant is typically successful only for small proteins. Oftentimes, refolding of large proteins leads to the formation of off-pathway intermediates and aggregation-prone conformations. Here we explore the hypothesis that refolding of large or otherwise complex proteins is challenging in part because in vitro refolding experiments do not replicate a key aspect of the intracellular environment in which proteins evolved to fold: vectorial appearance (and folding) of a protein.

Modeling native state ensembles and macromolecular complexes using hydrogen-deuterium exchange ensemble reweighting

Gaining a high-resolution understanding of the conformational landscape of proteins and protein complexes in solution remains a challenge. Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is an insightful approach in probing a protein's structural dynamics in solution. It reports on the structural environment of the backbone amide hydrogens of a protein in a time resolved manner. As such, HDX-MS has now been widely applied to study proteins within various biophysical perspectives including the characterization of native state ensembles of proteins, macromolecular interactions, protein-small molecule interactions or protein folding and unfolding studies.

Studying protein folding landscape using NMR relaxation measurements with pressure perturbation

Repeat proteins greatly simplify the identification of the sequence determinants for protein folding cooperativity. We use the leucine rich repeat (LRR) domain of the tumor suppressor pp32 as a model. It is composed of five LRR with a capping domain on each of its termini. Previous pressure-dependent NMR experiments of cavity-containing mutants of pp32 revealed that the intermediates populated during pressure-induced unfolding depend upon the position of the cavity. In order to further characterize the nature of these intermediates, we carried out NMR relaxation experiments, relaxation dispersion (CPMG) and chemical exchange saturation transfer (CEST) experiments.

In-cell NMR: a powerful approach for studying protein folding and for drug screening

In-cell NMR: a powerful approach for studying protein folding and for drug screening

Structural and functional adaptation in extremophilic microbial α-amylases.

Maintaining stable native conformation of a protein under a given ecological condition is the prerequisite for survival of organisms. Extremophilic bacteria and archaea have evolved to adapt under extreme conditions of temperature, pH, salt, and pressure. Molecular adaptations of proteins under these conditions are essential for their survival. These organisms have the capability to maintain stable, native conformations of proteins under extreme conditions. The enzymes produced by the extremophiles are also known as extremozyme, which are used in several industries. Stability and functionality of extremozymes under varying temperature, pH, and solvent conditions are the most desirable requirement of industry. α-Amylase is one of the most important enzymes used in food, pharmaceutical, textile, and detergent industries. This enzyme is produced by diverse microorganisms including various extremophiles. Therefore, understanding its stability is important from fundamental as well as an applied point of view. Each class of extremophiles has a distinctive set of dominant non-covalent interactions which are important for their stability. Static information obtained by comparative analysis of amino acid sequence and atomic resolution structure provides information on the prevalence of particular amino acids or a group of non-covalent interactions. Protein folding studies give the information about thermodynamic and kinetic stability in order to understand dynamic aspect of molecular adaptations. In this review, we have summarized information on amino acid sequence, structure, stability, and adaptability of α-amylases from different classes of extremophiles.

New insights into the folding-unfolding mechanism and conformations of cytochrome C.

Metalloproteins account for over one-third of all proteins in nature and play important roles in biological processes. The formation of the native structures of metalloproteins requires not only the correct folding of the polypeptide chains but also the proper incorporation of metal cofactors. Understanding the folding mechanism of metalloproteins has been challenging. Horse heart cytochrome C (cytc) is a classical model system for protein folding studies. Although a large number of ensemble studies have been carried out to characterize the folding mechanism of cytc, there is still a significant debate on the folding mechanism and the existence of the proposed “foldons”. Here, we used single-molecule optical tweezers to probe the mechanical folding–unfolding behaviors of cytc at the single-molecule level. By directly monitoring the folding and unfolding of holo-cytc, we revealed novel insights into the folding of cytc. Our results showed that the structural elements that are distant from the N- and C-termini can exist as a short-lived intermediate, a finding that contrasts with the general belief that the folding and packing of the N- and C-terminal helices are prerequisites for the folding of other structural elements in cytc. In addition, our results present strong evidence that apo-cytc, which has been long believed to be a random coil, is not a true random coil, and weak interactions within the unfolded polypeptide chain exist. Our results bring new insights into our understanding of the folding mechanisms of heme proteins as well as the role of heme in the folding process.

Variable-Temperature Native Mass Spectrometry for Studies of Protein Folding, Stabilities, Assembly, and Molecular Interactions.

The structures and conformational dynamics of proteins, protein complexes, and their noncovalent interactions with other molecules are controlled specifically by the Gibbs free energy (entropy and enthalpy) of the system. For some organisms, temperature is highly regulated, but the majority of biophysical studies are carried out at room, nonphysiological temperature. In this review, we describe variable-temperature electrospray ionization (vT-ESI) mass spectrometry (MS)-based studies with unparalleled sensitivity, dynamic range, and selectivity for studies of both cold- and heat-induced chemical processes. Such studies provide direct determinations of stabilities, reactivities, and thermodynamic measurements for native and non-native structures of proteins and protein complexes and for protein-ligand interactions. Highlighted in this review are vT-ESI-MS studies that reveal 40 different conformers of chymotrypsin inhibitor 2, a classic two-state (native → unfolded) unfolder, and thermochemistry for a model membrane protein system binding lipid and its regulatory protein.