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Biophysical Journal Feb 2023Temperature is a useful system variable to gather kinetic and thermodynamic information from proteins. Usually, free energy and the associated entropic and enthalpic...
Temperature is a useful system variable to gather kinetic and thermodynamic information from proteins. Usually, free energy and the associated entropic and enthalpic contributions are obtained by quantifying the conformational equilibrium based on melting experiments performed in bulk conditions. Such experiments are suitable only for those small single-domain proteins whose side reactions of irreversible aggregation are unlikely to occur. Here, we avoid aggregation by pulling single-protein molecules in a thermo-regulated optical tweezers. Thus, we are able to explore the temperature dependence of the thermodynamic and kinetic parameters of MJ0366 from Methanocaldococcus jannaschii at the single-molecule level. By performing force-ramp experiments between 2°C and 40°C, we found that MJ0366 has a nonlinear dependence of free energy with temperature and a specific heat change of 2.3 ± 1.2 kcal/molK. These thermodynamic parameters are compatible with a two-state unfolding/refolding mechanism for MJ0366. However, the kinetics measured as a function of the temperature show a complex behavior, suggesting a three-state folding mechanism comprising a high-energy intermediate state. The combination of two perturbations, temperature and force, reveals a high-energy species in the folding mechanism of MJ0366 not detected in force-ramp experiments at constant temperature.
Topics: Temperature; Protein Folding; Optical Tweezers; Thermodynamics; Entropy; Kinetics; Protein Denaturation
PubMed: 36587240
DOI: 10.1016/j.bpj.2022.12.034 -
The Journal of Biological Chemistry Aug 2020The 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...
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 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.
Topics: Enzyme Stability; Escherichia coli; Escherichia coli Proteins; Protein Folding; Protein Unfolding; Proteolysis; Ribonuclease H; Ribosomes
PubMed: 32527724
DOI: 10.1074/jbc.RA120.013909 -
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 -
ACS Nano Jul 2022Globular folded proteins are versatile nanoscale building blocks to create biomaterials with mechanical robustness and inherent biological functionality due to their...
Globular folded proteins are versatile nanoscale building blocks to create biomaterials with mechanical robustness and inherent biological functionality due to their specific and well-defined folded structures. Modulating the nanoscale unfolding of protein building blocks during network formation ( protein unfolding) provides potent opportunities to control the protein network structure and mechanics. Here, we control protein unfolding during the formation of hydrogels constructed from chemically cross-linked maltose binding protein using ligand binding and the addition of cosolutes to modulate protein kinetic and thermodynamic stability. Bulk shear rheology characterizes the storage moduli of the bound and unbound protein hydrogels and reveals a correlation between network rigidity, characterized as an increase in the storage modulus, and protein thermodynamic stability. Furthermore, analysis of the network relaxation behavior identifies a crossover from an unfolding dominated regime to an entanglement dominated regime. Control of protein unfolding and entanglement provides an important route to finely tune the architecture, mechanics, and dynamic relaxation of protein hydrogels. Such predictive control will be advantageous for future smart biomaterials for applications which require responsive and dynamic modulation of mechanical properties and biological function.
Topics: Hydrogels; Biocompatible Materials; Rheology; Proteins; Protein Unfolding
PubMed: 35731007
DOI: 10.1021/acsnano.2c02369 -
Molecular Cell Apr 2024Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded...
Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex rather than substrate recruitment. Experiments in yeast cells confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.
Topics: Adenosine Triphosphatases; Cell Cycle Proteins; Polyubiquitin; Proteasome Endopeptidase Complex; Proteolysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Valosin Containing Protein
PubMed: 38401542
DOI: 10.1016/j.molcel.2024.01.029 -
The Journal of Physical Chemistry. B Jun 2020Thermal protein unfolding resembles a global (two-state) phase transition. At the local scale, protein unfolding is, however, heterogeneous and probe dependent. Here, we...
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 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 CC 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.
Topics: Amino Acid Sequence; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Protein Unfolding; Thermodynamics
PubMed: 32392067
DOI: 10.1021/acs.jpcb.0c01230 -
Biochemical and Biophysical Research... Oct 2023The 26S proteasome is responsible for the unfolding and degradation of intracellular proteins in eukaryotes. A hexameric ring of ATPases (Rpt1-Rpt6) grabs onto...
The 26S proteasome is responsible for the unfolding and degradation of intracellular proteins in eukaryotes. A hexameric ring of ATPases (Rpt1-Rpt6) grabs onto substrates and unfolds them by pulling them through a central pore and translocating them into the 20S degradation chamber. A set of pore loops containing a so-called aromatic paddle motif in each Rpt subunit is believed to be important for the proteasome's ability to unfold and translocate substrates. Based on structural and mechanistic experiments, paddles from adjacent Rpt subunits, which are arrayed in a spiral staircase conformation, grip and pull on the substrate in a hand-over-hand type mechanism, disengaging at the bottom of the staircase and re-engaging at the top. We tested the contribution of the aromatic paddles to unfolding substrates of differing stabilities by mutating the paddles singly or in combination. For an easy-to-unfold substrate (a circular permutant of green fluorescent protein; GFP), mutations had little effect on degradation rates. For a substrate with moderate stability (enhanced GFP), there were modest effects of individual mutations on GFP unfolding rates, and alternating aromatic paddle mutants had a larger detrimental effect on unfolding than sequential mutants. For a more stable substrate (superfolder GFP), unfolding is overall slower, and multiple simultaneous mutations essentially prevent unfolding. Our results highlight the context-dependent need for grip during unfolding, support the hand-over-hand model for substrate unfolding and translocation, and suggest that for hard-to-unfold substrates, it is important to have simultaneous strong contacts to the substrate for unfolding to occur. The results also suggest a kinetic proofreading model, where substrates that cannot be easily unfolded are instead clipped, removing the initiation region and preventing futile unfolding attempts.
PubMed: 37591185
DOI: 10.1016/j.bbrc.2023.08.025 -
International Journal of Molecular... Mar 2021Using a number of optical techniques (interferometry, dynamic light scattering, and spectroscopy), denaturation of hen egg white lysozyme (HEWL) by treatment with a...
Using a number of optical techniques (interferometry, dynamic light scattering, and spectroscopy), denaturation of hen egg white lysozyme (HEWL) by treatment with a combination of dithiothreitol (DTT) and guanidine hydrochloride (GdnHCl) has been investigated. The denaturing solutions were selected so that protein denaturation occurred with aggregation (Tris-HCl pH = 8.0, 50 mM, DTT 30 mM) or without aggregation (Tris-HCl pH = 8.0, 50 mM, DTT 30 mM, GdnHCl 6 M) and can be evaluated after 60 min of treatment. It has been found that denatured by solution with 6 M GdnHCl lysozyme completely loses its enzymatic activity after 30 min and the size of the protein molecule increases by 1.5 times, from 3.8 nm to 5.7 nm. Denaturation without of GdnHCl led to aggregation with preserving about 50% of its enzymatic activity. Denaturation of HEWL was examined using interferometry. Previously, it has been shown that protein denaturation that occurs without subsequent aggregation leads to an increase in the refractive index (Δ ~ 4.5 × 10). This is most likely due to variations in the HEWL-solvent interface area. By applying modern optical techniques conjointly, it has been possible to obtain information on the nature of time-dependent changes that occur inside a protein and its hydration shell as it undergoes denaturation.
Topics: Animals; Chickens; Dithiothreitol; Guanidine; Muramidase; Protein Aggregates; Protein Unfolding; Spectrophotometry, Ultraviolet
PubMed: 33800175
DOI: 10.3390/ijms22052710 -
Role of freezing-induced myofibrillar protein denaturation in the generation of thaw loss: A review.Meat Science Aug 2022Formation of thaw loss cannot generally be avoided when meat is frozen and then thawed. Explanations have mainly focused on the damage to muscle fibers resulting from... (Review)
Review
Formation of thaw loss cannot generally be avoided when meat is frozen and then thawed. Explanations have mainly focused on the damage to muscle fibers resulting from ice crystallization and the freezing-induced denaturation of myofibrillar proteins, the latter of which has, however, not received much research focus. This review discusses the relationship between myofibrillar protein denaturation and water-holding capacity of meat in freezing-thawing with the aim to improve the understanding the relative importance of protein denaturation in the formation of thaw loss. The contribution of decreased pH and high ionic strength in the unfrozen water in freezing is emphasized and we hypothesize that these two factors are causing protein denaturation and conformational changes within muscle fibers, and consequently loss of water-holding capacity. Slow freezing produces more thaw loss than fast freezing, and this is discussed here in relation to the impacts on myofibrillar protein denaturation induced by the freezing rate.
Topics: Freezing; Meat; Protein Denaturation; Proteins; Water
PubMed: 35533633
DOI: 10.1016/j.meatsci.2022.108841 -
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