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There is still an insufficient understanding of how the characteristics of protein drugs are maintained in the solid state of lyophilizates, including aspects such as protein distances, local environment, and structural preservation. To this end, we evaluated protein folding and the molecules' nearest environment by electron paramagnetic resonance (EPR) spectroscopy. Double electron-electron resonance (DEER) probe distances of up to approximately 200 Å and is suitable to investigate protein folding, local concentration, and aggregation, whereas electron spin echo envelope modulation (ESEEM) allows the study of the near environment within approximately 10 Å of the spin label. We spin-labeled human serum albumin (HSA) and freeze-dried different concentrations with 100 g/L deuterated sucrose. DEER showed distinct local concentration behaviors for two folding states, directly correlating folding percentage with the interprotein distance, reaching 2 nm at an HSA concentration of 84 g/L. Interestingly, 50% of the HSA molecules showed partial structural perturbation already at 2.6 g/L, which corresponds to a molar ratio Suc/HSA of 7469. This percentage increased to 97% with an increase in the HSA concentration to 84 g/L. The degree of protein perturbation cannot be told, and no signs of unfolding are found after reconstitution. ESEEM demonstrated a higher sucrose concentration around the protein label compared to the HSA environment in highly concentrated sucrose solutions. The partial unfolding detected in DEER could lead to label exposure and explain the enhanced sucrose detection in the intimate shell. Our work provides new insights regarding sucrose enrichment in the nearest shell of proteins upon lyophilization. In addition, the results indicate substantial partial structural perturbation, even in the presence of enormous supplies of stabilizing sugars. Thus, pulse EPR spectroscopy allows additional understanding of the solid state of protein lyophilizates, which is complementary to SANS, FTIR, or ssNMR.

Protein folding and unfolding experiments are interpreted under the assumption of microscopic reversibility, that is, that at equilibrium one process is the reverse of the other. Single-domain proteins illustrate the validity of such an interpretation, although reversibility does not necessarily hold under the different conditions typically used for folding and unfolding experiments. In fact, more complex proteins, which often exhibit irreversible unfolding, are generally considered not amenable to folding kinetics studies. Here, the X11 PDZ1-PDZ2 tandem repeat allows us to reveal the different folding and unfolding pathways at play under different experimental conditions, thus reconciling the apparent contradiction between theory and experiment., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Biomolecular dynamics simulation is a fundamental technology for life sciences research, and its usefulness depends on its accuracy and efficiency 1-3 . Classical molecular dynamics simulation is fast but lacks chemical accuracy 4,5 . Quantum chemistry methods such as density functional theory can reach chemical accuracy but cannot scale to support large biomolecules 6 . Here we introduce an artificial intelligence-based ab initio biomolecular dynamics system (AI 2 BMD) that can efficiently simulate full-atom large biomolecules with ab initio accuracy. AI 2 BMD uses a protein fragmentation scheme and a machine learning force field 7 to achieve generalizable ab initio accuracy for energy and force calculations for various proteins comprising more than 10,000 atoms. Compared to density functional theory, it reduces the computational time by several orders of magnitude. With several hundred nanoseconds of dynamics simulations, AI 2 BMD demonstrated its ability to efficiently explore the conformational space of peptides and proteins, deriving accurate 3 J couplings that match nuclear magnetic resonance experiments, and showing protein folding and unfolding processes. Furthermore, AI 2 BMD enables precise free-energy calculations for protein folding, and the estimated thermodynamic properties are well aligned with experiments. AI 2 BMD could potentially complement wet-lab experiments, detect the dynamic processes of bioactivities and enable biomedical research that is impossible to conduct at present., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Most proteins fold during biosynthesis on the ribosome 1 , and co-translational folding energetics, pathways and outcomes of many proteins have been found to differ considerably from those in refolding studies 2-10 . The origin of this folding modulation by the ribosome has remained unknown. Here we have determined atomistic structures of the unfolded state of a model protein on and off the ribosome, which reveal that the ribosome structurally expands the unfolded nascent chain and increases its solvation, resulting in its entropic destabilization relative to the peptide chain in isolation. Quantitative 19 F NMR experiments confirm that this destabilization reduces the entropic penalty of folding by up to 30 kcal mol -1 and promotes formation of partially folded intermediates on the ribosome, an observation that extends to other protein domains and is obligate for some proteins to acquire their active conformation. The thermodynamic effects also contribute to the ribosome protecting the nascent chain from mutation-induced unfolding, which suggests a crucial role of the ribosome in supporting protein evolution. By correlating nascent chain structure and dynamics to their folding energetics and post-translational outcomes, our findings establish the physical basis of the distinct thermodynamics of co-translational protein folding., (© 2024. The Author(s).)

Optical tweezers (OT) have evolved into powerful single molecule force spectroscopy tools to investigate protein folding-unfolding dynamics. To stretch a protein of interest using OT, the protein must be flanked with two double stranded DNA (dsDNA) handles. However, coupling dsDNA handles to the protein is often of low yield, representing a bottleneck in OT experiments. Here, we report a handle-free, all-protein-based OT method for investigating protein folding/unfolding dynamics. In this new method, we employed disordered elastin-like polypeptides (ELPs) as a molecular linker and the mechanically stable cohesin-dockerin (Coh-Doc) pair as the prey-bait system to enable the efficient capture and stretching of individual protein molecules. This novel approach was validated by using model proteins NuG2 and RTX-v, yielding experimental results comparable to those obtained by using the dsDNA handle approach. This new method provides a streamlined and efficient OT approach to investigate the folding-unfolding dynamics of proteins at the single molecule level, thus expanding the toolbox of OT-based single molecule force spectroscopy.

Acylphosphatase (AcP) is an enzyme which catalyses the hydrolysis of acylphosphate. The binding with the phosphate ion (Pi) assumes significance in preserving both the stability and enzymatic activity of AcP. While previous studies using single molecule force spectroscopy explored the mechanical properties of AcP, the influence of Pi on its folding and unfolding dynamic behaviors remains unexplored. In this work, using stable magnetic tweezers, we measured and compared the force-dependent folding and unfolding rates of AcP in the Tris buffer and phosphate buffer within a force range from 2 pN to 40 pN. We found that Pi exerts no discernible effect on the folding dynamics but consistently decreases the force-dependent unfolding rate of AcP by a constant ratio across the entire force spectrum. The free energy landscapes of AcP in the absence and presence of Pi are constructed. Our results reveal that Pi selectively binds to the native state of AcP, stabilizing it and suggesting the general properties of specific ligand-receptor interactions.

Protein folding is a fascinating, not fully understood phenomenon in biology. Molecular dynamics (MD) simulations are an invaluable tool to study conformational changes in atomistic detail, including folding and unfolding processes of proteins. However, the accuracy of the conformational ensembles derived from MD simulations inevitably relies on the quality of the underlying force field in combination with the respective water model. Here, we investigate protein folding, unfolding, and misfolding of fast-folding proteins by examining different force fields with their recommended water models, i.e., ff14SB with the TIP3P model and ff19SB with the OPC model. To this end, we generated long conventional MD simulations highlighting the perks and pitfalls of these setups. Using Markov state models, we defined kinetically independent conformational substates and emphasized their distinct characteristics, as well as their corresponding state probabilities. Surprisingly, we found substantial differences in thermodynamics and kinetics of protein folding, depending on the combination of the protein force field and water model, originating primarily from the different water models. These results emphasize the importance of carefully choosing the force field and the respective water model as they determine the accuracy of the observed dynamics of folding events. Thus, the findings support the hypothesis that the water model is at least equally important as the force field and hence needs to be considered in future studies investigating protein dynamics and folding in all areas of biophysics.

Interprotein interactions between the partially unfolded states of γ D-crystallin ( γ D-crys) protein are known to cause cataracts. Therefore, understanding the unfolding pathways of native γ D-crys is extremely crucial to delineate their aggregation mechanism. In this study, we have performed extensive all-atom Molecular Dynamics simulations with explicit solvent to understand the role of the critical residues that drive the stability of the motifs and domains of γ D-crys in its wild type and mutant forms. Our findings show that while the individual motifs of wild type are not stable in the native form, the individual domains remain structurally stable at 425K. This enhanced stability of the domain was attributed to the hydrophobic interactions between the motifs. Single and double point mutations of the domains with negatively charged aspartic and glutamic acid amino acid residues (I3E, W42D, W42E, I3D/W42D, I3E/W42E, and L92D/W157D) decreases the structural stability, leading to unfolding of individual domains of γ D-crys. We believe that our study sheds light on the weakest links of γ D-crys, along with the role of interactions stabilizing the domains. Further, this study bolsters and provides a better understanding of the domain swapping mechanism of aggregation of γ D-crys.Communicated by Ramaswamy H. Sarma.

The small organic molecules, known as osmolytes being ubiquitously present in different cell types, affect protein folding, stability and aggregation. However, it is unknown how the osmolytes affect the nanomechanical unfolding behavior of protein domain. Here, we show the osmolyte-dependent mechanical unfolding properties of protein titin immunoglobulin-27 (I27) domain using an atomic force microscopy (AFM)-based single-molecule force spectroscopy. We found that amines and methylamines improved the mechanical stability of I27 domain, whereas polyols had no effect. Interestingly, glycine betaine (GB) or trimethylamine-N-oxide (TMAO) increased the average unfolding force of the protein domain. The kinetic parameters analyzed at single-molecule level reveal that stabilizing effect of osmolytes is due to a decrease in the unfolding rate constant of I27, which was confirmed by molecular dynamics simulations. Our study reveals different effects that diverse osmolytes have on the mechanical properties of the protein, and suggests the potential use of osmolytes in modulating the mechanical stability of proteins required for various nano-biotechnological applications., Competing Interests: Declaration of competing interest The authors declare that there is no competing interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Single molecule techniques are particularly well suited for investigating the processes of protein folding and chaperone assistance. However, current assays provide only a limited perspective on the various ways in which the cellular environment can influence the folding pathway of a protein. In this study, a single molecule mechanical interrogation assay is developed and used to monitor protein unfolding and refolding within a cytosolic solution. This allows to test the cumulative topological effect of the cytoplasmic interactome on the folding process. The results reveal a stabilization against forced unfolding for partial folds, which are attributed to the protective effect of the cytoplasmic environment against unfolding and aggregation. This research opens the possibility of conducting single molecule molecular folding experiments in quasi-biological environments., (© 2023 The Authors. Advanced Biology published by Wiley-VCH GmbH.)

Most proteins fold into characteristic three-dimensional structures. The rate of folding and unfolding varies widely and can be affected by variations in proteins. We developed a novel machine-learning-based method for the prediction of the folding rate effects of amino acid substitutions in two-state folding proteins. We collected a data set of experimentally defined folding rates for variants and used them to train a gradient boosting algorithm starting with 1161 features. Two predictors were designed. The three-class classifier had, in blind tests, specificity and sensitivity ranging from 0.324 to 0.419 and from 0.256 to 0.451, respectively. The other tool was a regression predictor that showed a Pearson correlation coefficient of 0.525. The error measures, mean absolute error and mean squared error, were 0.581 and 0.603, respectively. One of the previously presented tools could be used for comparison with the blind test data set, our method called PON-Fold showed superior performance on all used measures. The applicability of the tool was tested by predicting all possible substitutions in a protein domain. Predictions for different conformations of proteins, open and closed forms of a protein kinase, and apo and holo forms of an enzyme indicated that the choice of the structure had a large impact on the outcome. PON-Fold is freely available.

Cystatin superfamily members, by virtue of their thiol protease regulatory properties, show involvement in myriad physiological processes important for survival and well-being. The current study involves urea-induced denaturation of a novel variant of the cystatin superfamily, rai seed cystatin (RSC), employing a variety of biophysical assays in order to characterize different folding intermediates generated on unfolding. Urea as a denaturant presented the passage of RSC through a series of events resulting in the loss of RSC functional capability, accompanied by changes in the archetype at secondary and tertiary structural levels, as evident from protease inhibitory, UV absorption, and intrinsic fluorescence assays, respectively. ANS fluorescence also revealed routing of RSC through discrete multiple sub-states thus presenting the generation of intermediate states somewhat close to the pre-molten globule and/or molten globule forms of RSC. Furthermore, far-UV circular dichroism analysis revealed a concentration-dependent gradual loss in typical -helical RSC peaks, indicating a nearly 50 % loss in secondary structural elements around 5 M urea treatment. The study also reports the possible role of glycerol in the refolding and/or reactivation of the urea unfolded RSC form. Glycerol presented itself as a potent structural stabilizer as it assisted in the refolding and reactivation of the unfolded RSC in a dosage-dependent manner, concomitantly paving the way for unravelling the mechanistic approach involved in the phenomenon, which can facilitate future studies., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest in this work., (Copyright © 2023. Published by Elsevier B.V.)

Detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the lack of a comprehensive characterization of the aggregates, which are typically heterogeneous. Here we designed a reporter chaperone substrate, MLucV, composed of a stress-labile luciferase flanked by stress-resistant fluorescent domains, which upon denaturation formed a discrete population of small aggregates. Combining Förster resonance energy transfer and enzymatic activity measurements provided unprecedented details on the aggregated, unfolded, Hsp70-bound and native MLucV conformations. The Hsp70 mechanism first involved ATP-fueled disaggregation and unfolding of the stable pre-aggregated substrate, which stretched MLucV beyond simply unfolded conformations, followed by native refolding. The ATP-fueled unfolding and refolding action of Hsp70 on MLucV aggregates could accumulate native MLucV species under elevated denaturing temperatures highly adverse to the native state. These results unambiguously exclude binding and preventing of aggregation from the non-equilibrium mechanism by which Hsp70 converts stable aggregates into metastable native proteins., (© 2022. The Author(s).)

A highly bioluminescent protein, NanoLuc (Nluc), has seen numerous applications in biological assays since its creation. We recently engineered a NanoLuc polyprotein that showed high bioluminescence but displayed a strong misfolding propensity after mechanical unfolding. Here, we present our single-molecule force spectroscopy (SMFS) studies by atomic force microscopy (AFM) and steered molecular dynamics (SMD) simulations on two new hybrid protein constructs comprised of Nluc and I91 titin domains, I91-I91-Nluc-I91-I91-I91-I91 (I91 2 -Nluc-I91 4 ) and I91-Nluc-I91-Nluc-I91-Nluc-I91, to characterize the unfolding behavior of Nluc in detail and to further investigate its misfolding properties that we observed earlier for the I91 2 -Nluc 3 -I91 2 construct. Our SMFS results confirm that Nluc's unfolding proceeds similarly in all constructs; however, Nluc's refolding differs in these constructs, and its misfolding is minimized when Nluc is monomeric or separated by I91 domains. Our simulations on monomeric Nluc, Nluc dyads, and Nluc triads pinpointed the origin of its mechanical stability and captured interesting unfolding intermediates, which we also observed experimentally.

The performance of a protein depends on its correct folding to the final functional native form. Hence, understanding the process of protein folding has remained an important field of research for the scientific community for the past five decades. Two important intermediate states, namely, wet molten globule (WMG) and dry molten globule (DMG), have emerged as critical milestones during protein folding-unfolding reactions. While much has been discussed about WMGs as a common unfolding intermediate, the evidence for DMGs has remained elusive owing to their near-native features, which makes them difficult to probe using global structural probes. This Review puts together the available literature and new evidence on DMGs to give a broader perspective on the universality of DMGs and discuss their significance in protein folding, function, and disease.

Understanding how high hydrostatic pressure affects biomacromolecular interaction is important for deciphering the molecular mechanisms by which organisms adapt to live at the bottom of the ocean. The relative effect of hydrostatic pressure on the rates of folding/unfolding reactions is defined by the volumetric properties of the transition state ensemble relative to the folded and unfolded states. All-atom structure-based molecular dynamics simulations combined with quantitative computational protocol to compute volumes from three-dimensional coordinates allow volumetric mapping of protein folding landscape. This, is turn, provides qualitative understanding of the effects of hydrostatic pressure on energy landscape of proteins. The computational results for six different proteins are directly benchmark against experimental data and show an excellent agreement. Both experiments and computation show that the transition-state ensemble volume appears to be in-between the folded and unfolded state volumes, and thus the hydrostatic pressure accelerates protein unfolding.

The spliceosome protein U1A is a prototype case of the RNA recognition motif (RRM) ubiquitous in biological systems. The in vitro kinetics of the chemical denaturation of U1A indicate that the unfolding of U1A is a two-state process but takes place via high energy channeling and a malleable transition state, an interesting variation of typical two-state behavior. Molecular dynamics (MD) simulations have been applied extensively to the study of two-state unfolding and folding of proteins and provide an opportunity to obtain a theoretical account of the experimental results and a molecular model for the transition state ensemble. We describe herein all-atom MD studies including explicit solvent of up to 100 ns on the thermal unfolding (UF) of U1A and 13 mutants. Multiple MD UF trajectories are carried out to ensure accuracy and reproducibility. A vector representation of the MD unfolding process in RMSD space is obtained and used to calculate a free energy landscape for U1A unfolding. A corresponding MD simulation and free energy landscape for the protein CI2, well known to follow a simple two state folding/unfolding model, is provided as a control. The results indicate that the unfolding pathway on the MD calculated free energy landscape of U1A shows a markedly extended transition state compared with that of CI2. The MD results support the interpretation of the observed chevron plots for U1A in terms of a high energy, channel-like transition state. Analysis of the MDUF structures shows that the transition state ensemble involves microstates with most of the RRM secondary structure intact but expanded by ~14% with respect to the radius of gyration. Comparison with results on a prototype system indicates that the transition state involves an ensemble of molten globule structures and extends over the region of ~1-35 ns in the trajectories. Additional MDUF simulations were carried out for 13 U1A mutants, and the calculated φ-values show close accord with observed results and serve to validate our methodology.

Molecular chaperones in cells constantly monitor and bind to exposed hydrophobicity in newly synthesized proteins and assist them in folding or targeting to cellular membranes for insertion. However, proteins can be misfolded or mistargeted, which often causes hydrophobic amino acids to be exposed to the aqueous cytosol. Again, chaperones recognize exposed hydrophobicity in these proteins to prevent nonspecific interactions and aggregation, which are harmful to cells. The chaperone-bound misfolded proteins are then decorated with ubiquitin chains denoting them for proteasomal degradation. It remains enigmatic how molecular chaperones can mediate both maturation of nascent proteins and ubiquitination of misfolded proteins solely based on their exposed hydrophobic signals. In this review, we propose a dynamic ubiquitination and deubiquitination model in which ubiquitination of newly synthesized proteins serves as a "fix me" signal for either refolding of soluble proteins or retargeting of membrane proteins with the help of chaperones and deubiquitinases. Such a model would provide additional time for aberrant nascent proteins to fold or route for membrane insertion, thus avoiding excessive protein degradation and saving cellular energy spent on protein synthesis. Also see the video abstract here: https://youtu.be/gkElfmqaKG4., (© 2022 Wiley Periodicals LLC.)

NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and separate detection of the folded and unfolded state as well as possible equilibrium intermediates. This allows a detailed view on the state and cooperativity of folding of the protein of interest and the correct interpretation of subsequent experiments. Here we summarize in detail practical and theoretical aspects of such experiments. Certain pitfalls can be avoided, and meaningful simplification can be made during the analysis. Especially a good understanding of the NMR exchange regime and relaxation properties of the system of interest is beneficial. We show by a global analysis of signals of the folded and unfolded state of GB1 how accurate values of unfolding can be extracted and what limits different NMR detection and unfolding methods. E.g. commonly used exchangeable amides can lead to a systematic under determination of the thermodynamic protein stability. We give several perspectives of how to deal with more complex proteins and how the knowledge about protein stability at residue resolution helps to understand protein properties under crowding conditions, during phase separation and under high pressure., (© 2022. The Author(s).)

Understanding protein folding is crucial for protein sciences. The conformational spaces and energy landscapes of cold (unfolded) protein states, as well as the associated transitions, are hardly explored. Furthermore, it is not known how structure relates to the cooperativity of cold transitions, if cold and heat unfolded states are thermodynamically similar, and if cold states play important roles for protein function. We created the cold unfolding 4-helix bundle DCUB1 with a de novo designed bipartite hydrophilic/hydrophobic core featuring a hydrogen bond network which extends across the bundle in order to study the relative importance of hydrophobic versus hydrophilic protein-water interactions for cold unfolding. Structural and thermodynamic characterization resulted in the discovery of a complex energy landscape for cold transitions, while the heat unfolded state is a random coil. Below ∼0 °C, the core of DCUB1 disintegrates in a largely cooperative manner, while a near-native helical content is retained. The resulting cold core-unfolded state is compact and features extensive internal dynamics. Below -5 °C, two additional cold transitions are seen, that is, (i) the formation of a water-mediated, compact, and highly dynamic dimer, and (ii) the onset of cold helix unfolding decoupled from cold core unfolding. Our results suggest that cold unfolding is initiated by the intrusion of water into the hydrophilic core network and that cooperativity can be tuned by varying the number of core hydrogen bond networks. Protein design has proven to be invaluable to explore the energy landscapes of cold states and to robustly test related theories.

Protein quality control systems are crucial for cellular function and organismal health. At present, most known protein quality control systems are multicomponent machineries that operate via ATP-regulated interactions with non-native proteins to prevent aggregation and promote folding 1 , and few systems that can broadly enable protein folding by a different mechanism have been identified. Moreover, proteins that contain the extensively charged poly-Asp/Glu (polyD/E) region are common in eukaryotic proteomes 2 , but their biochemical activities remain undefined. Here we show that DAXX, a polyD/E protein that has been implicated in diverse cellular processes 3-10 , possesses several protein-folding activities. DAXX prevents aggregation, solubilizes pre-existing aggregates and unfolds misfolded species of model substrates and neurodegeneration-associated proteins. Notably, DAXX effectively prevents and reverses aggregation of its in vivo-validated client proteins, the tumour suppressor p53 and its principal antagonist MDM2. DAXX can also restore native conformation and function to tumour-associated, aggregation-prone p53 mutants, reducing their oncogenic properties. These DAXX activities are ATP-independent and instead rely on the polyD/E region. Other polyD/E proteins, including ANP32A and SET, can also function as stand-alone, ATP-independent molecular chaperones, disaggregases and unfoldases. Thus, polyD/E proteins probably constitute a multifunctional protein quality control system that operates via a distinctive mechanism., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute-protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute-protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, [Formula: see text], and an effective correlation time, τ c The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute-protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the [Formula: see text] values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state., Competing Interests: The authors declare no competing interest.

Native mass spectrometry (MS) enables the study of intact proteins as well as noncovalent protein-protein and protein-ligand complexes in their biological state. In this work, we present the application of a Waters desorption electrospray ionization (DESI) source with a prototype spray emitter for rapid surface measurements of folded and native protein structures. A comparison of DESI spray solvent shows that adding 50% methanol to 200 mM ammonium acetate solution does not reduce its performance in preserving folded protein structures. Instead, improved signal-to-noise (S/N) ratio is obtained, and less adducted peaks are detected by using this uncommon native MS solvent system. The standard DESI design with an inlet tube allows optimization of sampling temperature conditions to improve desolvation and therefore S/N ratio. Furthermore, tuning the inlet temperature enables the control and study of unfolding behavior of proteins from surface samples. The optimized condition for native DESI has been applied to several selected proteins and protein complexes with the molecular weight ranging from 8.6 to 66.4 kDa. Ions of folded proteins with narrow charge state distribution (CSD), or peaks showing noncovalent-bond-assembled intact protein complexes, are observed in the spectra. Evidence for the structural refolding of denatured proteins and protein complexes sampled with native solvent highlights the need for care when interpreting DESI native MS data, particularly for proteins with stable native structures.

Unfolding and refolding of multidomain proteins under force have yet to be recognized as a major mechanism of function for proteins in vivo. In this review, we discuss the inherent properties of multidomain proteins under a force vector from a structural and functional perspective. We then characterize three main systems where multidomain proteins could play major roles through mechanical unfolding: muscular contraction, cellular mechanotransduction, and bacterial adhesion. We analyze how key multidomain proteins for each system can produce a gain-of-function from the perspective of a fine-tuned quantized response, a molecular battery, delivery of mechanical work through refolding, elasticity tuning, protection and exposure of cryptic sites, and binding-induced mechanical changes. Understanding how mechanical unfolding and refolding affect function will have important implications in designing mechano-active drugs against conditions such as muscular dystrophy, cancer, or novel antibiotics., (© 2020 Federation of European Biochemical Societies.)

Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature T m , should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X-ray crystallography and cryo-electron microscopy, screening for small-molecule binding partners and comparison of effects of point mutations., (© 2020 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Single molecule force spectroscopy (SMFS) has emerged since the past few years as a prominent set of techniques, within the broader field of atomic force microscopy (AFM), for the study of interactions and binding forces of individual protein molecules. Since force spectroscopy measures the behavior of a molecule when stretching or torsional mechanical force is applied, it can be an excellent tool in the hands of researchers who study protein folding and misfolding mechanisms, by reverse engineering the forced unfolding. Such studies could be of crucial importance in the field of protein-related diseases. In this review we wish to provide a glimpse at SMFS concept and recent advances, paired with the protein misfolding issue in neurodegenerative diseases., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Downhill folding has been defined as a unique thermodynamic process involving a conformations ensemble that progressively loses structure with the decrease of protein stability. Downhill folders are estimated to be rather rare in nature as they miss an energetically substantial folding barrier that can protect against aggregation and proteolysis. We have previously demonstrated that the prokaryotic zinc finger protein Ros87 shows a bipartite folding/unfolding process in which a metal binding intermediate converts to the native structure through a delicate barrier-less downhill transition. Significant variation in folding scenarios can be detected within protein families with high sequence identity and very similar folds and for the same sequence by varying conditions. For this reason, we here show, by means of DSC, CD and NMR, that also in different pH and ionic strength conditions Ros87 retains its partly downhill folding scenario demonstrating that, at least in metallo-proteins, the downhill mechanism can be found under a much wider range of conditions and coupled to other different transitions. We also show that mutations of Ros87 zinc coordination sphere produces a different folding scenario demonstrating that the organization of the metal ion core is determinant in the folding process of this family of proteins.

High-hydrostatic pressure is an alternative perturbation method that can be used to destabilize globular proteins. Generally perfectly reversible, pressure exerts local effects on regions or domains of a protein containing internal voids, contrary to heat or chemical denaturant that destabilize protein structures uniformly. When combined with NMR spectroscopy, high pressure (HP) allows one to monitor at a residue-level resolution the structural transitions occurring upon unfolding and to determine the kinetic properties of the process. The use of HP-NMR has long been hampered by technical difficulties. Owing to the recent development of commercially available high-pressure sample cells, HP-NMR experiments can now be routinely performed. This review summarizes recent advances of HP-NMR techniques for the characterization at a quasi-atomic resolution of the protein folding energy landscape.

Atomistic simulations can provide valuable, experimentally-verifiable insights into protein folding mechanisms, but existing ab initio simulation methods are restricted to only the smallest proteins due to severe computational speed limits. The folding of larger proteins has been studied using native-centric potential functions, but such models omit the potentially crucial role of non-native interactions. Here, we present an algorithm, entitled DBFOLD, which can predict folding pathways for a wide range of proteins while accounting for the effects of non-native contacts. In addition, DBFOLD can predict the relative rates of different transitions within a protein's folding pathway. To accomplish this, rather than directly simulating folding, our method combines equilibrium Monte-Carlo simulations, which deploy enhanced sampling, with unfolding simulations at high temperatures. We show that under certain conditions, trajectories from these two types of simulations can be jointly analyzed to compute unknown folding rates from detailed balance. This requires inferring free energies from the equilibrium simulations, and extrapolating transition rates from the unfolding simulations to lower, physiologically-reasonable temperatures at which the native state is marginally stable. As a proof of principle, we show that our method can accurately predict folding pathways and Monte-Carlo rates for the well-characterized Streptococcal protein G. We then show that our method significantly reduces the amount of computation time required to compute the folding pathways of large, misfolding-prone proteins that lie beyond the reach of existing direct simulation. Our algorithm, which is available online, can generate detailed atomistic models of protein folding mechanisms while shedding light on the role of non-native intermediates which may crucially affect organismal fitness and are frequently implicated in disease., Competing Interests: The authors have declared that no competing interests exist.

Molecular dynamics simulations are an invaluable tool to characterize the dynamic motions of proteins in atomistic detail. However, the accuracy of models derived from simulations inevitably relies on the quality of the underlying force field. Here, we present an evaluation of current non-polarizable and polarizable force fields (AMBER ff14SB, CHARMM 36m, GROMOS 54A7, and Drude 2013) based on the long-standing biophysical challenge of protein folding. We quantify the thermodynamics and kinetics of the β-hairpin formation using Markov state models of the fast-folding mini-protein CLN025. Furthermore, we study the (partial) folding dynamics of two more complex systems, a villin headpiece variant and a WW domain. Surprisingly, the polarizable force field in our set, Drude 2013, consistently leads to destabilization of the native state, regardless of the secondary structure element present. All non-polarizable force fields, on the other hand, stably characterize the native state ensembles in most cases even when starting from a partially unfolded conformation. Focusing on CLN025, we find that the conformational space captured with AMBER ff14SB and CHARMM 36m is comparable, but the ensembles from CHARMM 36m simulations are clearly shifted toward disordered conformations. While the AMBER ff14SB ensemble overstabilizes the native fold, CHARMM 36m and GROMOS 54A7 ensembles both agree remarkably well with experimental state populations. In addition, GROMOS 54A7 also reproduces experimental folding times most accurately. Our results further indicate an over-stabilization of helical structures with AMBER ff14SB. Nevertheless, the presented investigations strongly imply that reliable (un)folding dynamics of small proteins can be captured in feasible computational time with current additive force fields.

The health of a cell depends on accurate translation and proper protein folding, whereas misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the nascent chain energy landscape. However, quantifying these environmental effects is challenging because ribosomal proteins and rRNA preclude most spectroscopic measurements of protein energetics. Here, we have applied two gel-based approaches, pulse proteolysis and force-profile analysis, to probe the folding and unfolding pathways of RNase H (RNH) nascent chains stalled on the prokaryotic ribosome in vitro We found that ribosome-stalled RNH has an increased unfolding rate compared with free RNH. Because protein stability is related to the ratio of the unfolding and folding rates, this increase completely accounts for the observed change in protein stability and indicates that the folding rate is unchanged. Using arrest peptide-based force-profile analysis, we assayed the force generated during the folding of RNH on the ribosome. Surprisingly, we found that population of the RNH folding intermediate is required to generate sufficient force to release a stall induced by the SecM stalling sequence and that readthrough of SecM directly correlates with the stability of the RNH folding intermediate. Together, these results imply that the folding pathway of RNH is unchanged on the ribosome. Furthermore, our findings indicate that the ribosome promotes RNH unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of RNH misfolding and assist in folding fidelity., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Jensen et al.)

Determining the temperature at which the thermal unfolding of a protein starts becoming irreversible is relevant for many areas of protein research. Until now, published methods cannot determine, within a reasonable time frame and with moderate sample consumption, the exposure temperature that starts causing irreversible protein unfolding. We present modulated scanning fluorimetry (MSF) and share a software (MSF Analyzer), which can be used to derive nonreversibility curves of thermal protein unfolding from a series of incremental temperature cycles performed on only 10 μL samples, consuming as low as a few micrograms of protein. Further processing of the data can yield the onset temperature that starts causing nonreversible protein unfolding. The MSF method is based on the hardware of the already existing nanoDSF technology and can be applied to dozens of samples simultaneously. Here, we use MSF to study how solution pH affects the reversibility of thermal protein unfolding of several model proteins to show that the nonreversibility onset temperature ( T ) is a unique biophysical parameter, providing orthogonal information from thermal protein denaturation data and insights into the validity of thermal unfolding analysis in the context of equilibrium thermodynamics. We also show that MSF can be used to study enzyme stability after exposure to high temperatures. Besides, we demonstrate that protein thermal unfolding and nonreversibility can be affected in different ways upon modifications like PEG-ylation or labeling with fluorescent dyes. Finally, we show that MSF can be used to study the effect of various protein interactions on thermal protein unfolding reversibility. With the diverse examples in this work, we reveal how MSF can provide orthogonal information from thermal denaturation experiments that can bring benefits to various areas of protein research. The MSF Analyzer software is available at https://github.com/CoriolisPharmaResearch/MSFAnalyser.nr ) is a unique biophysical parameter, providing orthogonal information from thermal protein denaturation data and insights into the validity of thermal unfolding analysis in the context of equilibrium thermodynamics. We also show that MSF can be used to study enzyme stability after exposure to high temperatures. Besides, we demonstrate that protein thermal unfolding and nonreversibility can be affected in different ways upon modifications like PEG-ylation or labeling with fluorescent dyes. Finally, we show that MSF can be used to study the effect of various protein interactions on thermal protein unfolding reversibility. With the diverse examples in this work, we reveal how MSF can provide orthogonal information from thermal denaturation experiments that can bring benefits to various areas of protein research. The MSF Analyzer software is available at https://github.com/CoriolisPharmaResearch/MSFAnalyser.

Knots are remarkable topological features in nature. The presence of knots in crystallographic structures of proteins have stimulated considerable research to determine the kinetic and thermodynamic consequences of threading a polypeptide chain. By mechanically manipulating MJ0366, a small single domain protein harboring a shallow trefoil knot, we allow the protein to refold from either the knotted or the unknotted denatured state to characterize the free energy profile associated to both folding pathways. By comparing the stability of the native state with reference to the knotted and unknotted denatured state we find that knotting the polypeptide chain of MJ0366 increase the folding energy barrier in a magnitude close to the energy cost of forming a knot randomly in the denatured state. These results support that a protein knot can be formed during a single cooperative step of folding but occurs at the expenses of a large increment on the free energy barrier.

Thermal protein unfolding resembles a global (two-state) phase transition. At the local scale, protein unfolding is, however, heterogeneous and probe dependent. Here, we consider local order parameters defined by the local curvature and torsion of the protein main chain. Because chemical shifts (CS's) measured by NMR spectroscopy are extremely sensitive to the local atomic environment, CS has served as a local probe of thermal unfolding of proteins by varying the position of the atomic isotope along the amino acid sequence. The variation of the CS of each C α atom along the sequence as a function of the temperature defines a local heat-induced denaturation curve. We demonstrate that these local heat-induced denaturation curves mirror the local protein nativeness defined by the free energy landscape of the local curvature and torsion of the protein main chain described by the C α - virtual bonds. Comparison between molecular dynamics simulations and CS data of the gpW protein demonstrates that some local native states defined by the local curvature and torsion of the main chain, mainly located in secondary structures, are coupled to each other whereas others, mainly located in flexible protein segments, are not. Consequently, CS's of some residues are faithful reporters of global protein unfolding, with heat-induced denaturation curves similar to the average global one, whereas other residues remain silent about the protein unfolded state. For the latter, the local deformation of the protein main chain, characterized by its local curvature and torsion, is not cooperatively coupled to global unfolding.α virtual bonds. Comparison between molecular dynamics simulations and CS data of the gpW protein demonstrates that some local native states defined by the local curvature and torsion of the main chain, mainly located in secondary structures, are coupled to each other whereas others, mainly located in flexible protein segments, are not. Consequently, CS's of some residues are faithful reporters of global protein unfolding, with heat-induced denaturation curves similar to the average global one, whereas other residues remain silent about the protein unfolded state. For the latter, the local deformation of the protein main chain, characterized by its local curvature and torsion, is not cooperatively coupled to global unfolding.

Hyperpolarized water can be a valuable aid in protein NMR, leading to amide group 1 H polarizations that are orders of magnitude larger than their thermal counterparts. Suitable procedures can exploit this to deliver 2D 1 H- 15 N correlations with good resolution and enhanced sensitivity. These enhancements depend on the exchange rates between the amides and the water, thereby yielding diagnostic information about solvent accessibility. This study applied this "HyperW" method to four proteins exhibiting a gamut of exchange behaviors: PhoA (350-471) , an unfolded 122-residue fragment; barstar, a fully folded ribonuclease inhibitor; R17, a 13.3 kDa system possessing folded and unfolded forms under slow interconversion; and drkN SH3, a protein domain whose folded and unfolded forms interchange rapidly and with temperature-dependent population ratios. For PhoA4 (350-471) HyperW sensitivity enhancements were ≥300×, as expected for an unfolded protein sequence. Though fully folded, barstar also exhibited substantial enhancements; these, however, were not uniform and, according to CLEANEX experiments, reflected the solvent-exposed residues. R17 showed the expected superposition of ≥100-fold enhancements for its unfolded form, coexisting with more modest enhancements for their folded counterparts. Unexpected, however, was the behavior of drkN SH3, for which HyperW enhanced the unfolded but, surprisingly, enhanced even more certain folded protein sites. These preferential enhancements were repeatedly and reproducibly observed. A number of explanations-including three-site exchange magnetization transfers between water and the unfolded and folded states; cross-correlated relaxation processes from hyperpolarized "structural" waters and labile side-chain protons; and the possibility that faster solvent exchange rates characterize certain folded sites over their unfolded counterparts-are considered to account for them.

Backbone circularization is an effective technique for protein stabilization. Here, we investigated the effect of a connector, an engineered segment that connects two protein termini, on the conformational stability of previously designed circularized variants of granulocyte colony-stimulating factor (G-CSF). Heat tolerance and chemical denaturation analyses revealed that aggregation resistance and thermodynamic stability of the circularized variants were superior to those of linear G-CSF. Crystal structure and molecular dynamics (MD) simulation of the most thermodynamically stable variant (C166) revealed a high number of intramolecular hydrogen bonds in both the connector region and Helix D adjacent to the connector region in the folded structure. MD simulations and theoretical calculations involving different force fields indicated a reduction in the main chain entropy of C166 in the unfolded state and increase in the intramolecular hydrogen bond energy of C166 in the folded structure. Although backbone circularization is usually considered to alter chain entropy of the unfolded state, the data indicated that it could also improve the conformational enthalpy of the folded state. Further structural examination of the connector region confirmed that protein design based on a statistical analysis of local structures is an effective approach for predicting an optimum connector length to improve the conformational stability of backbone-circularized proteins. Protein design using backbone circularization with an optimum connector length will be useful for the development of effective and safe protein therapeutics. DATABASE: Structural data are available in Protein Data Bank under the accession number 5ZO6., (© 2019 Federation of European Biochemical Societies.)

Mechanical processes are involved at many stages of the development of living cells, and often external forces applied to a biomolecule result in its unfolding. Although our knowledge of the unfolding mechanisms and the magnitude of the forces involved has evolved, the role that water molecules play in the mechanical unfolding of biomolecules has not yet been fully elucidated. To this end, we investigated with steered molecular dynamics simulations the mechanical unfolding of dystrophin's spectrin repeat 1 and related the changes in the protein's structure to the ordering of the surrounding water molecules. Our results indicate that upon mechanically induced unfolding of the protein, the solvent molecules become more ordered and increase their average number of hydrogen bonds. In addition, the unfolded structures originating from mechanical pulling expose an increasing amount of the hydrophobic residues to the solvent molecules, and the uncoiled regions adapt a convex surface with a small radius of curvature. As a result, the solvent molecules reorganize around the protein's small protrusions in structurally ordered waters that are characteristic of the so-called "small-molecule regime," which allows water to maintain a high hydrogen bond count at the expense of an increased structural order. We also determined that the response of water to structural changes in the protein is localized to the specific regions of the protein that undergo unfolding. These results indicate that water plays an important role in the mechanically induced unfolding of biomolecules. Our findings may prove relevant to the ever-growing interest in understanding macromolecular crowding in living cells and their effects on protein folding, and suggest that the hydration layer may be exploited as a means for short-range allosteric communication., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The correlations between the logarithm of the unfolding rate of 108 proteins and their structural parameters were calculated. We showed that there is a good correlation between the logarithm of folding and unfolding rates (0.79) and protein stability and unfolding rate (0.79). Thus, the faster the protein folds, the faster it unfolds. Folding and unfolding rates are higher for the proteins with two-state kinetics, in comparison with the proteins with multi-state kinetics. At the same time, two-state bacterial proteins folds and unfolds two orders of magnitude faster than two-state eukaryotic proteins, and multi-state bacterial proteins folds and unfolds slower than multi-state eukaryotic proteins. Despite the fact that the folding rates of thermophilic and mesophilic proteins are close, the unfolding rates of thermophilic proteins is about two orders of magnitude lower than for mesophilic proteins. The correlation between unfolding rate and stability of thermophilic proteins is high (0.90). We also found that the unfolding rate correlates with such structural parameters as: size of the protein, radius of the cross-section, logarithm of absolute contact order, and radius of gyration. This information will be useful for engineering and designing new proteins with desired properties.

Characterizing folding and complex formation of biomolecules provides a view into their thermodynamics, kinetics and folding pathways. Deciphering kinetic intermediates is particularly important because they can often be targeted by drugs. The key advantage of native mass spectrometry over conventional methods that monitor a single observable is its ability to identify and quantify coexisting species. Here, we show the design of a temperature-jump electrospray source for mass spectrometry that allows one to perform fast kinetics experiments (0.16-32 s) at different temperatures (10-90 °C). The setup allows recording of both folding and unfolding kinetics by using temperature jumps from high to low, and low to high, temperatures. Six biological systems, ranging from peptides to proteins to DNA complexes, exemplify the use of this device. Using temperature-dependent experiments, the folding and unfolding of a DNA triplex are studied, providing detailed information on its thermodynamics and kinetics.

Protein dynamics covers multiple spatiotemporal scale processes, among which slow motions, not much understood even though they are underlying protein folding and protein functions. Protein slow motions are associated with structural heterogeneity, short-lived and poorly populated conformations, hard to detect individually. In addition, they involve collective motions of many atoms, not easily tracked by simulation and experimental devices. Here we propose a biophysical approach, coupling geometrical nanoconfinement and broadband dielectric spectroscopy (BDS), which distinguishes protein conformations by their respective molecular dynamics. In particular, protein-unfolding intermediates, usually poorly populated in macroscopic solutions are detected. The protein dynamics is observed under unusual conditions (sample nanoconfinement and dehydration) highlighting the robustness of protein structure and protein dynamics to a variety of conditions consistent with protein sustainability. The protein dielectric signals evolve with the temperature of thermal treatments indicating sensitivity to atomic and molecular interaction changes triggered by the protein thermal unfolding. As dipole fluctuations depend on both collective large-scale motions and local motions, the approach offers a prospect to track in-depth unfolding events.

In this work, we measured the millisecond residue specific protein folding and unfolding dynamics in E. coli cells for two protein GB3 mutants using NMR. The results show that the protein folding and unfolding dynamics in cells is different from that in buffer. Through a two-site exchange model, it is shown that both the population and the exchange rate are changed by the E. coli cellular environment. Further investigation suggests that the change is likely due to the quinary interaction with crowded molecules in the cell. Our work underlines the importance of cellular environment to protein folding kinetics and thermodynamics although this environmental effect may not be large enough to change the protein structure.

Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Background: Human aldose reductase (hAR) is the first and rate-limiting enzyme of the polyol pathway. For the development of secondary complications of diabetes in chronic hyperglycemic conditions, one of the critical factors is the increased flux of glucose through the polyol pathway.  Due to this clinical implication, hAR attracted considerable attention from the drug discovery perspective. In spite of extensive characterization in the context of biochemical and structural aspects, we know very little about the unfolding behavior of hAR. This study reports equilibrium unfolding studies of hAR. Methods: We carried out thermal denaturation and chemical-induced equilibrium unfolding studies of hAR monitored by circular dichroism and fluorescence spectroscopy.  Results: Thermal denaturation studies presented a classical picture of two-state unfolding from native to the denatured state. The data was used to derive thermodynamic parameters and study the thermostability of hAR. Chemical induced equilibrium unfolding studies led us to discover an intermediate state, which gets populated at 3.5-4.0 M and 0.7-2.0 M of urea and GuHCl, respectively. Thermodynamic parameters derived from chemical-induced unfolding are in agreement with those obtained from thermal denaturation of hAR. Conclusion: This study revealed that aldose reductase unfolds from native to the unfolded state via an intermediate. Assessment of the thermodynamic stability of native, intermediate, and unfolded states shows that significant energy barriers separate these states, which ensures the cooperativity of unfolding. As hAR functions in cells that are under osmotic and oxidative stress, these in vitro findings may have implications for its native conformation under the physiological state., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Sekhon G and Singh R.)

Protein folding/unfolding is a complicated process that defies high-resolution characterization by experimental methods. As an alternative, atomistic molecular dynamics simulations are now routinely employed to elucidate and magnify the accompanying conformational changes and the role of solvent in the folding process. However, the level of detail necessary to map the process at high spatial-temporal resolution provides an overwhelming amount of data. As more and better tools are developed for analysis of these large data sets and validation of the simulations, one is still left with the problem of visualizing the results in ways that provide insight into the folding/unfolding process. While viewing and interrogating static crystal structures has become commonplace, more and different approaches are required for dynamic, interconverting, unfolding, and refolding proteins. Here we review a variety of approaches, ranging from straightforward to complex and unintuitive for multiscale analysis and visualization of protein folding and unfolding., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Intrinsically disordered proteins (IDPs) are extensively involved in dynamic signaling processes which require a high association rate and a high dissociation rate for rapid binding/unbinding events and at the same time a sufficient high affinity for specific recognition. Although the coupled folding-binding processes of IDPs have been extensively studied, it is still impossible to predict whether an unfolded protein is suitable for molecular signaling via coupled folding-binding. In this work, we studied the interplay between intrinsic folding mechanisms and coupled folding-binding process for unfolded proteins through molecular dynamics simulations. We first studied the folding process of three representative IDPs with different folded structures, that is, c-Myb, AF9, and E3 rRNase. We found the folding free energy landscapes of IDPs are downhill or show low barriers. To further study the influence of intrinsic folding mechanism on the binding process, we modulated the folding mechanism of barnase via circular permutation and simulated the coupled folding-binding process between unfolded barnase permutant and folded barstar. Although folding of barnase was coupled to target binding, the binding kinetics was significantly affected by the intrinsic folding free energy barrier, where reducing the folding free energy barrier enhances binding rate up to two orders of magnitude. This accelerating effect is different from previous results which reflect the effect of structure flexibility on binding kinetics. Our results suggest that coupling the folding of an unfolded protein with no/low folding free energy barrier with its target binding may provide a way to achieve high specificity and rapid binding/unbinding kinetics simultaneously., (© 2018 Wiley Periodicals, Inc.)

The formulation of novel therapeutic proteins is a challenging task which aims at finding formulation conditions that will minimize protein degradation during long-term storage. One particularly important and difficult-to-predict protein degradation pathway is the so-called non-native aggregation. The qualitative and quantitative prediction of the latter has been a subject of extensive research over the past two decades. An increasing body of evidence shows that the widely-used short-term biophysical techniques cannot accurately rank formulation conditions in order of their effect on the aggregation during long-term storage of some therapeutic proteins, e.g. monoclonal antibodies. Here we suggest a novel approach for the selection of formulation conditions that will suppress the formation of protein aggregates during long-term storage. We postulate that conditions (i.e. pH, buffer type, ionic strength) that reduce the isothermal aggregation of various denaturant-induced partially folded protein species will be conditions that impede protein aggregation during long-term storage. To test our hypothesis, we developed an isothermal microdialysis-based unfolding/refolding assay, named ReFOLD, which we use to induce moderate aggregation of partially folded proteins. Next, we assessed the relative monomer yield after isothermal unfolding/refolding of two monoclonal antibodies, each formulated in 12 different conditions. Using the proposed approach, we were able to accurately rank the formulations in order of their effect on the amount of protein aggregates detected after storage for 12 months at 4 °C and 25 °C, while widely-used stability-indicating parameters like protein melting and aggregation onset temperatures failed to provide accurate predictive formulation rankings., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cdc48/p97 is an essential and highly conserved AAA+ ATPase that uses its protein-unfoldase activity to extract ubiquitinated polypeptides from macromolecular complexes and membranes. This motor has also been implicated in protein-degradation pathways, yet its exact role in acting upstream of the 26S proteasome remains elusive. Ubiquitinated proteins destined for degradation by the proteasome require an unstructured initiation region to engage with the proteasomal translocation machinery, and Cdc48 was proposed to generate these unfolded segments, yet direct evidence has been missing. Here, we used an in vitro reconstituted system to demonstrate the collaboration of Cdc48 and the 26S proteasome from S . cerevisiae in degrading ubiquitinated, well-folded proteins that lack unstructured segments. Our data indicate that a critical role for Cdc48 in the ubiquitin-proteasome system is to create flexible initiation regions in compact substrates that otherwise would be refractory to engagement and degradation by the proteasome., Competing Interests: The authors declare no competing interests.

The marginal stability of globular proteins in the cell is determined by the balance between excluded volume effect and soft interactions. Quinary interactions are a type of soft interactions involved in intracellular organisation and known to have stabilising or destabilising effects on globular proteins. Recent studies suggest that globular proteins have structural flexibility, exhibiting more than one functional state. Here, we propose that the quinary-induced destabilisation can be sufficient to produce functional partially unfolded states of globular proteins. The biological relevance of this mechanism is explored, involving intracellular phase separation and regulatory stress response mechanisms., (© 2018 Federation of European Biochemical Societies.)

Since the proposal of Anfinsen's thermodynamic hypothesis in 1963, our understanding of protein folding and dynamics has gained significant appreciation of its nuance and complexity. Intrinsically disordered proteins, chameleonic sequences, morpheeins, and metamorphic proteins have broadened the protein folding paradigm. Here, we discuss noncanonical protein folding patterns, with an emphasis on metamorphic proteins, and we review known metamorphic proteins that occur naturally and that have been engineered in the laboratory. Finally, we discuss research areas surrounding metamorphic proteins that are primed for future exploration, including evolution, drug discovery, and the quest for previously unrecognized metamorphs. As we enter an age where we are capable of complex bioinformatic searches and de novo protein design, we are primed to search for previously unrecognized metamorphic proteins and to design our own metamorphs to act as targeted, switchable drugs; biosensors; and more.

Direct tracking of protein structural dynamics during folding-unfolding processes is important for understanding the roles of hierarchic structural factors in the formation of functional proteins. Using cytochrome c (cyt c) as a platform, we investigated its structural dynamics during folding processes triggered by local environmental changes (i.e., pH or heme iron center oxidation/spin/ligation states) with time-resolved X-ray solution scattering measurements. Starting from partially unfolded cyt c, a sudden pH drop initiated by light excitation of a photoacid caused a structural contraction in microseconds, followed by active site restructuring and unfolding in milliseconds. In contrast, the reduction of iron in the heme via photoinduced electron transfer did not affect conformational stability at short timescales (<1 ms), despite active site coordination geometry changes. These results demonstrate how different environmental perturbations can change the nature of interaction between the active site and protein conformation, even within the same metalloprotein, which will subsequently affect the folding structural dynamics.