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Biochemistry Aug 2020The properties of enzymes packaged within the coat protein shell of virus-like particles (VLPs) were studied to provide a comprehensive assessment of such factors. Such...
The properties of enzymes packaged within the coat protein shell of virus-like particles (VLPs) were studied to provide a comprehensive assessment of such factors. Such entrainment did not seem to perturb enzyme function, but it did significantly enhance enzyme stability against several denaturing stimuli such as heat, organic solvents, and chaotropic agents. This improvement in performance was found to be general and independent of the number of independent subunits required and of the number of catalytically active enzymes packaged. Packaged enzymes were found by measurements of intrinsic tryptophan fluorescence to retain some of their native folded structure even longer than their catalytic activity, suggesting that protein folding is a significant component of the observed catalytic benefits. While we are unable to distinguish between kinetic and thermodynamic effects - including inhibition of enzyme unfolding, acceleration of refolding, and biasing of folding equilibria - VLP packaging appears to represent a useful general strategy for the stabilization of enzymes that operate on diffusible substrates and products.
Topics: Biocatalysis; Enzyme Stability; Enzymes; Leviviridae; Protein Unfolding; Solvents; Virion
PubMed: 32786888
DOI: 10.1021/acs.biochem.0c00435 -
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 -
Analytica Chimica Acta Feb 2023Unclear issues in protein studies include but not limited to the stability and denaturation mechanism in the presence of denaturants. Herein, we report a dynamic...
Unclear issues in protein studies include but not limited to the stability and denaturation mechanism in the presence of denaturants. Herein, we report a dynamic monitoring approach based on nanopore single-molecule biosensor, which can detect the protein's folding and unfolding transitions by recording a nanopore ionic current. When gradually increasing the concentration of denaturant guanidine hydrochloride (GdmCl), sensitive responses were observed with lysozyme unfolding. The emergence of the featured biphasic-pulse demonstrated the existence of a stable intermediate. It was the first time to experimentally confirm the dynamic equilibrium between the intermediate and the native states at single molecule level, therefore consolidating the standpoint of lysozyme denaturation process following the three-state model. Additionally, we got more insights into the conformation about the intermediate as globular-like structure, larger gyration radius, and enhanced positive charge density. We considered that the manner of denaturant toward lysozyme adopts the "direct" model based on stronger electrostatic and van der Waals forces. Nanopore biosensor exhibited excellent sensitivity with a low detection concentration of 280 pM and reproducibility in analysing the folding intermediate of lysozyme.
Topics: Protein Folding; Protein Denaturation; Muramidase; Nanopores; Reproducibility of Results; Guanidine; Kinetics; Thermodynamics; Protein Conformation
PubMed: 36697181
DOI: 10.1016/j.aca.2023.340830 -
Inorganic Chemistry Jan 2022Human carbonic anhydrase II (HCA) is a robust metalloprotein and an excellent biological model system to study the thermodynamics of metal ion coordination. Apo-HCA...
Human carbonic anhydrase II (HCA) is a robust metalloprotein and an excellent biological model system to study the thermodynamics of metal ion coordination. Apo-HCA binds one zinc ion or two copper ions. We studied these binding processes at five temperatures (15-35 °C) using isothermal titration calorimetry, yielding thermodynamic parameters corrected for pH and buffer effects. We then sought to identify binding-induced structural changes. Our data suggest that binding at the active site organizes 6-8 residues; however, copper binding near the N-terminus results in a net of 6-7 residues. This surprising destabilization was confirmed using circular dichroism and protein stability measurements. Metal binding induced unfolding may represent an important regulatory mechanism, but it may be easily missed by NMR and X-ray crystallography. Thus, in addition to highlighting a highly novel binding-induced unfolding event, we demonstrate the value of calorimetry for studying the structural implications of metal binding.
Topics: Binding Sites; Calorimetry; Carbonic Anhydrase II; Carbonic Anhydrase Inhibitors; Coordination Complexes; Copper; Humans; Ions; Protein Unfolding; Zinc
PubMed: 34989562
DOI: 10.1021/acs.inorgchem.1c03271 -
Journal of the American Chemical Society Jul 2019In this work, we measured the millisecond residue specific protein folding and unfolding dynamics in cells for two protein GB3 mutants using NMR. The results show that...
In this work, we measured the millisecond residue specific protein folding and unfolding dynamics in 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 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.
Topics: DNA Glycosylases; Escherichia coli; Escherichia coli Proteins; Magnetic Resonance Spectroscopy; Mutation; Protein Conformation; Protein Folding; Protein Unfolding
PubMed: 31305080
DOI: 10.1021/jacs.9b04435 -
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 -
Proceedings of the National Academy of... Oct 2019Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously...
Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified "invisible" folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å.
Topics: Bacteriophage T4; Muramidase; Nuclear Magnetic Resonance, Biomolecular; Pressure; Protein Conformation; Protein Denaturation; Protein Folding; Proteins
PubMed: 31570587
DOI: 10.1073/pnas.1911181116 -
Methods in Molecular Biology (Clifton,... 2024Single-molecule atomic force microscopy (AFM) allows capturing the conformational dynamics of an individual molecule under force. In this chapter, we describe a protocol...
Single-molecule atomic force microscopy (AFM) allows capturing the conformational dynamics of an individual molecule under force. In this chapter, we describe a protocol for conducting a protein nanomechanical experiment using AFM, covering both the force-extension and force-clamp modes. Combined, these experiments provide an integrated vista of the molecular mechanisms-and their associated kinetics-underpinning the mechanical unfolding and refolding of individual proteins when exposed to mechanical load.
Topics: Microscopy, Atomic Force; Proteins; Nanotechnology; Mechanical Phenomena; Molecular Conformation; Protein Folding; Protein Unfolding
PubMed: 37824012
DOI: 10.1007/978-1-0716-3377-9_16 -
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 -
Analytical Chemistry Mar 2020In modern biochemistry, protein stability and ligand interactions are of high interest. These properties are often studied with methods requiring labeled biomolecules,...
In modern biochemistry, protein stability and ligand interactions are of high interest. These properties are often studied with methods requiring labeled biomolecules, as the existing methods utilizing luminescent external probes suffer from low sensitivity. Currently available label-free technologies, e.g., thermal shift assays, circular dichroism, and differential scanning calorimetry, enable studies on protein unfolding and protein-ligand interactions (PLI). Unfortunately, the required micromolar protein concentration increases the costs and predisposes these methods for spontaneous protein aggregation. Here, we report a time-resolved luminescence method for protein unfolding and PLI detection with nanomolar sensitivity. The Protein-Probe method is based on highly luminescent europium chelate-conjugated probe, which is the key component in sensing the hydrophobic regions exposed to solution after protein unfolding. With the same Eu-probe, we also demonstrate ligand-interaction induced thermal stabilization with model proteins. The developed Protein-Probe method provides a sensitive approach overcoming the problems of the current label-free methodologies.
Topics: Ligands; Models, Molecular; Protein Binding; Protein Denaturation; Protein Stability; Protein Structure, Secondary; Proteins; Temperature; Transition Temperature
PubMed: 32013400
DOI: 10.1021/acs.analchem.9b05712