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ACS Nano Jul 2021Hierarchical assemblies of proteins exhibit a wide-range of material properties that are exploited both in nature and by artificially by humankind. However, little is...
Hierarchical assemblies of proteins exhibit a wide-range of material properties that are exploited both in nature and by artificially by humankind. However, little is understood about the importance of protein unfolding on the network assembly, severely limiting opportunities to utilize this nanoscale transition in the development of biomimetic and bioinspired materials. Here we control the force lability of a single protein building block, bovine serum albumin (BSA), and demonstrate that protein unfolding plays a critical role in defining the architecture and mechanics of a photochemically cross-linked native protein network. The internal nanoscale structure of BSA contains "molecular reinforcement" in the form of 17 covalent disulphide "nanostaples", preventing force-induced unfolding. Upon addition of reducing agents, these nanostaples are broken rendering the protein force labile. Employing a combination of circular dichroism (CD) spectroscopy, small-angle scattering (SAS), rheology, and modeling, we show that stapled protein forms reasonably homogeneous networks of cross-linked fractal-like clusters connected by an intercluster region of folded protein. Conversely, protein unfolding results in more heterogeneous networks of denser fractal-like clusters connected by an intercluster region populated by unfolded protein. In addition, gelation-induced protein unfolding and cross-linking in the intercluster region changes the hydrogel mechanics, as measured by a 3-fold enhancement of the storage modulus, an increase in both the loss ratio and energy dissipation, and markedly different relaxation behavior. By controlling the protein's ability to unfold through nanoscale (un)stapling, we demonstrate the importance of unfolding in defining both network architecture and mechanics, providing insight into fundamental hierarchical mechanics and a route to tune biomaterials for future applications.
Topics: Hydrogels; Protein Unfolding; Biocompatible Materials; Serum Albumin, Bovine; Rheology
PubMed: 34214394
DOI: 10.1021/acsnano.1c00353 -
Biophysical Journal Jan 2016The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments...
The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale.
Topics: Bacterial Proteins; Biomechanical Phenomena; Elasticity; Kinetics; Mechanical Phenomena; Models, Molecular; Peptides; Protein Conformation; Protein Unfolding; Ribonucleases; Thermodynamics
PubMed: 26745410
DOI: 10.1016/j.bpj.2015.11.015 -
PloS One 2021Molecular basis of protein stability at different temperatures is a fundamental problem in protein science that is substantially far from being accurately and...
Molecular basis of protein stability at different temperatures is a fundamental problem in protein science that is substantially far from being accurately and quantitatively solved as it requires an explicit knowledge of the temperature dependence of folding free energy of amino acid residues. In the present study, we attempted to gain insights into the thermodynamic stability of SazCA and its implications on protein folding/unfolding. We report molecular dynamics simulations of water solvated SazCA in a temperature range of 293-393 K to study the relationship between the thermostability and flexibility. Our structural analysis shows that the protein maintains the highest structural stability at 353 K and the protein conformations are highly flexible at temperatures above 353 K. Larger exposure of hydrophobic surface residues to the solvent medium for conformations beyond 353 K were identified from H-bond analysis. Higher number of secondary structure contents exhibited by SazCA at 353 K corroborated the conformations at 353 K to exhibit the highest thermal stability. The analysis of thermodynamics of protein stability revealed that the conformations that denature at higher melting temperatures tend to have greater maximum thermal stability. Our analysis shows that 353 K conformations have the highest melting temperature, which was found to be close to the experimental optimum temperature. The enhanced protein stability at 353 K due the least value of heat capacity at unfolding suggested an increase in folding. Comparative Gibbs free energy analysis and funnel shaped energy landscape confirmed a transition in folding/unfolding pathway of SazCA at 353 K.
Topics: Bacteria; Bacterial Proteins; Carbonic Anhydrases; Enzyme Stability; Molecular Dynamics Simulation; Protein Unfolding
PubMed: 33857217
DOI: 10.1371/journal.pone.0249866 -
Proceedings of the National Academy of... Feb 2016Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments....
Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments. Unfolding rate, [Formula: see text], as a function of force f, obtained in single-molecule pulling experiments on src SH3 domain, exhibits upward curvature on a [Formula: see text] plot. Similar observations were reported for other proteins for the unfolding rate [Formula: see text]. These findings imply unfolding in these single-domain proteins involves a switch in the pathway as f or [Formula: see text] is increased from a low to a high value. We provide a unified theory demonstrating that if [Formula: see text] as a function of a perturbation (f or [Formula: see text]) exhibits upward curvature then the underlying energy landscape must be strongly multidimensional. Using molecular simulations we provide a structural basis for the switch in the pathways and dramatic shifts in the transition-state ensemble (TSE) in src SH3 domain as f is increased. We show that a single-point mutation shifts the upward curvature in [Formula: see text] to a lower force, thus establishing the malleability of the underlying folding landscape. Our theory, applicable to any perturbation that affects the free energy of the protein linearly, readily explains movement in the TSE in a β-sandwich (I27) protein and single-chain monellin as the denaturant concentration is varied. We predict that in the force range accessible in laser optical tweezer experiments there should be a switch in the unfolding pathways in I27 or its mutants.
Topics: Amino Acid Sequence; Animals; Biomechanical Phenomena; Chickens; Models, Molecular; Molecular Sequence Data; Movement; Mutant Proteins; Protein Unfolding; Thermodynamics; src Homology Domains
PubMed: 26818842
DOI: 10.1073/pnas.1515730113 -
ACS Nano Jul 2017Single-molecule studies of protein folding hold keys to unveiling protein folding pathways and elusive intermediate folding states-attractive pharmaceutical targets....
Single-molecule studies of protein folding hold keys to unveiling protein folding pathways and elusive intermediate folding states-attractive pharmaceutical targets. Although conventional single-molecule approaches can detect folding intermediates, they presently lack throughput and require elaborate labeling. Here, we theoretically show that measurements of ionic current through a nanopore containing a protein can report on the protein's folding state. Our all-atom molecular dynamics (MD) simulations show that the unfolding of a protein lowers the nanopore ionic current, an effect that originates from the reduction of ion mobility in proximity to a protein. Using a theoretical model, we show that the average change in ionic current produced by a folding-unfolding transition is detectable despite the orientational and conformational heterogeneity of the folded and unfolded states. By analyzing millisecond-long all-atom MD simulations of multiple protein transitions, we show that a nanopore ionic current recording can detect folding-unfolding transitions in real time and report on the structure of folding intermediates.
Topics: Databases, Protein; Ion Transport; Molecular Dynamics Simulation; Nanopores; Protein Conformation; Protein Folding; Protein Unfolding; Proteins
PubMed: 28693322
DOI: 10.1021/acsnano.7b02718 -
Protein Science : a Publication of the... Dec 2022Protein folding and unfolding is a complex process, underscored by the many proteotoxic diseases associated with misfolded proteins. Mapping pathways from a native...
Protein folding and unfolding is a complex process, underscored by the many proteotoxic diseases associated with misfolded proteins. Mapping pathways from a native structure to an unfolded protein or vice versa, identifying the intermediates, and defining the role of sequence and structure en route remain outstanding problems in the field. It is even more challenging to capture the events at atomistic resolution. X-ray diffraction has so far been used to understand how urea interacts with and unfolds two stable globular proteins. Here, we present the case study on PSMD10 , a prototype for a moderately stable, non-globular repeat protein, long and rigid, with its termini located at either end. We define structural changes in the time dimension using low urea concentrations to arrive at the following conclusions. (a) Unfolding is rapidly initiated at the C-terminus, slowly at the N-terminus, and proceeds inwards from both ends. (b) C-terminus undergoes an α to 3 helix transition, representing the structure of a potential early unfolding intermediate before disorder sets in. (c) Distinct and progressive changes in the electrostatic landscape of PSMD10 were observed, indicative of conformational changes in the seemingly inflexible motif involved in protein-protein interaction. We believe this unique study will open up the field for better and bolder queries and increase the choice of model proteins for a better understanding of the challenging problems of protein folding, protein interactions, protein degradation, and diseases associated with misfolding.
Topics: Protein Denaturation; Urea; Ankyrin Repeat; Protein Folding; X-Ray Diffraction; Protein Conformation; Protein Unfolding
PubMed: 36382986
DOI: 10.1002/pro.4515 -
Protein Science : a Publication of the... Dec 2020Protein aggregation is one of the most critical processes affecting protein solubility in various contexts-from protein therapeutics formulation to protein diseases. In...
Protein aggregation is one of the most critical processes affecting protein solubility in various contexts-from protein therapeutics formulation to protein diseases. In general, time-dependent changes in protein solubility are complex kinetically driven processes that often involve a triggering event that consists of a protein unfolding/misfolding followed by the assembling of aggregation-competent protein species. In this study, we have examined the relation between stability and time-dependent solubility of the recombinant human antibody light chain, hLC, which was found to form renal tubular casts in the multiple myeloma patient. To analyze the aggregation quantitatively, the hLC stability and protein solubility assays were performed in vitro at elevated temperatures. A differential acceleration of the processes at high temperatures enabled us to dissect observed kinetics of irreversible hLC unfolding and aggregation. We find that for hLC these processes have different molecularity and activation energy barriers. While the irreversible unfolding of hLC is a unimolecular step with a substantial activation energy barrier of 260 kJ/mol, the aggregation is rate-limited by the bimolecular reaction, which is characterized by a lower activation energy barrier of 40 kJ/mol. By the combination of experimental assays at different temperatures, different protein concentrations and kinetic modeling using ordinary differential equations, we were able to extrapolate time-dependent protein solubility to temperatures where both unfolding and aggregation processes are strongly kinetically coupled. Our study enables mechanism-based evaluation and interpretation of different physico-chemical factors contributing to the hLC unfolding and aggregation and their effect on the formation of extracellular protein deposits.
Topics: Humans; Immunoglobulin Light Chains; Kinetics; Multiple Myeloma; Neoplasm Proteins; Protein Aggregates; Protein Unfolding
PubMed: 33030218
DOI: 10.1002/pro.3968 -
Current Opinion in Structural Biology Feb 2017Extracellular matrix fibrils of fibronectin (FN) are highly elastic, and are typically stretched three to four times their relaxed length. The mechanism of stretching... (Review)
Review
Extracellular matrix fibrils of fibronectin (FN) are highly elastic, and are typically stretched three to four times their relaxed length. The mechanism of stretching has been controversial, in particular whether it involves tension-induced unfolding of FNIII domains. Recent studies have found that ∼5pN is the threshold isometric force for unfolding various protein domains. FNIII domains should therefore not be unfolded until the tension approaches 5pN. Integrins have been reported to generate forces ranging from 1 to >50pN, but I argue that studies reporting 1-2pN are the most convincing. This is not enough to unfold FNIII domains. Even if domains were unfolded, 2pN would only extend the worm-like-chain to about twice the length of the folded domain. Overall I conclude that stretching FN matrix fibrils involves primarily the compact to extended conformational change of FN dimers, with minimal contribution from unfolding FNIII domains.
Topics: Fibronectins; Integrins; Mechanical Phenomena; Protein Domains; Protein Refolding; Protein Unfolding
PubMed: 28038331
DOI: 10.1016/j.sbi.2016.12.002 -
Solid State Nuclear Magnetic Resonance Apr 2019In this article we give an overview over the use of DNP-enhanced solid-state NMR spectroscopy for the investigation of unfolded, disordered and misfolded proteins. We... (Review)
Review
In this article we give an overview over the use of DNP-enhanced solid-state NMR spectroscopy for the investigation of unfolded, disordered and misfolded proteins. We first provide an overview over studies in which DNP spectroscopy has successfully been applied for the structural investigation of well-folded amyloid fibrils formed by short peptides as well as full-length proteins. Sample cooling to cryogenic temperatures often leads to severe line broadening of resonance signals and thus a loss in resolution. However, inhomogeneous line broadening at low temperatures provides valuable information about residual dynamics and flexibility in proteins, and, in combination with appropriate selective isotope labeling techniques, inhomogeneous linewidths in disordered proteins or protein regions may be exploited for evaluation of conformational ensembles. In the last paragraph we highlight some recent studies where DNP-enhanced MAS-NMR-spectroscopy was applied to the study of disordered proteins/protein regions and inhomogeneous sample preparations.
Topics: Humans; Nuclear Magnetic Resonance, Biomolecular; Protein Stability; Protein Unfolding; Proteins; Temperature
PubMed: 30641444
DOI: 10.1016/j.ssnmr.2018.12.003 -
Analytical Chemistry Dec 2020Native ion mobility-mass spectrometry (IM-MS) is capable of revealing much that remains unknown within the structural proteome, promising such information on refractory...
Native ion mobility-mass spectrometry (IM-MS) is capable of revealing much that remains unknown within the structural proteome, promising such information on refractory protein targets. Here, we report the development of a unique drift tube IM-MS (DTIM-MS) platform, which combines high-energy source optics for improved collision induced unfolding (CIU) experiments and an electromagnetostatic cell for electron capture dissociation (ECD). We measured a series of high precision collision cross section (CCS) values for protein and protein complex ions ranging from 6-1600 kDa, exhibiting an average relative standard deviation (RSD) of 0.43 ± 0.20%. Furthermore, we compare our CCS results to previously reported DTIM values, finding strong agreement across similarly configured instrumentation (average RSD of 0.82 ± 0.73%), and systematic differences for DTIM CCS values commonly used to calibrate traveling-wave IM separators (-3% average RSD). Our CIU experiments reveal that the modified DTIM-MS instrument described here achieves enhanced levels of ion activation when compared with any previously reported IM-MS platforms, allowing for comprehensive unfolding of large multiprotein complex ions as well as interplatform CIU comparisons. Using our modified DTIM instrument, we studied two protein complexes. The enhanced CIU capabilities enable us to study the gas phase stability of the GroEL 7-mer and 14-mer complexes. Finally, we report CIU-ECD experiments for the alcohol dehydrogenase tetramer, demonstrating improved sequence coverage by combining ECD fragmentation integrated over multiple CIU intermediates. Further improvements for such native top-down sequencing experiments were possible by leveraging IM separation, which enabled us to separate and analyze CID and ECD fragmentation simultaneously.
Topics: Electrons; Mass Spectrometry; Protein Unfolding; Proteins
PubMed: 33166123
DOI: 10.1021/acs.analchem.0c03372