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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 -
Applied Microbiology and Biotechnology Mar 2021Overexpression of recombinant proteins in Escherichia coli results in misfolded and non-active protein aggregates in the cytoplasm, so-called inclusion bodies (IB). In... (Review)
Review
Overexpression of recombinant proteins in Escherichia coli results in misfolded and non-active protein aggregates in the cytoplasm, so-called inclusion bodies (IB). In recent years, a change in the mindset regarding IBs could be observed: IBs are no longer considered an unwanted waste product, but a valid alternative to produce a product with high yield, purity, and stability in short process times. However, solubilization of IBs and subsequent refolding is necessary to obtain a correctly folded and active product. This protein refolding process is a crucial downstream unit operation-commonly done as a dilution in batch or fed-batch mode. Drawbacks of the state-of-the-art include the following: the large volume of buffers and capacities of refolding tanks, issues with uniform mixing, challenging analytics at low protein concentrations, reaction kinetics in non-usable aggregates, and generally low re-folding yields. There is no generic platform procedure available and a lack of robust control strategies. The introduction of Quality by Design (QbD) is the method-of-choice to provide a controlled and reproducible refolding environment. However, reliable online monitoring techniques to describe the refolding kinetics in real-time are scarce. In our view, only monitoring and control of re-folding kinetics can ensure a productive, scalable, and versatile platform technology for re-folding processes. For this review, we screened the current literature for a combination of online process analytical technology (PAT) and modeling techniques to ensure a controlled refolding process. Based on our research, we propose an integrated approach based on the idea that all aspects that cannot be monitored directly are estimated via digital twins and used in real-time for process control. KEY POINTS: • Monitoring and a thorough understanding of refolding kinetics are essential for model-based control of refolding processes. • The introduction of Quality by Design combining Process Analytical Technology and modeling ensures a robust platform for inclusion body refolding.
Topics: Inclusion Bodies; Kinetics; Protein Folding; Protein Refolding; Recombinant Proteins; Technology
PubMed: 33598720
DOI: 10.1007/s00253-021-11151-y -
The FEBS Journal Mar 2022Folding stability is a crucial feature of protein evolution and is essential for protein functions. Thus, the comprehension of protein folding mechanisms represents an...
Folding stability is a crucial feature of protein evolution and is essential for protein functions. Thus, the comprehension of protein folding mechanisms represents an important complement to protein structure and function, crucial to determine the structural basis of protein misfolding. In this context, thermal unfolding studies represent a useful tool to get a molecular description of the conformational transitions governing the folding/unfolding equilibrium of a given protein. Here, we report the thermal folding/unfolding pathway of VEGFR1D2, a member of the immunoglobulin superfamily by means of a high-resolution thermodynamic approach that combines differential scanning calorimetry with atomic-level unfolding monitored by NMR. We show how VEGFR1D2 folding is driven by an oxidatively induced disulfide pairing: the key event in the achievement of its functional structure is the formation of a small hydrophobic core that surrounds a disulfide bridge. Such a 'folding nucleus' induces the cooperative transition to the properly folded conformation supporting the hypothesis that a disulfide bond can act as a folding nucleus that eases the folding process.
Topics: Calorimetry, Differential Scanning; Circular Dichroism; Disulfides; Humans; Protein Denaturation; Protein Folding; Proteins; Thermodynamics
PubMed: 34689403
DOI: 10.1111/febs.16246 -
Proceedings of the National Academy of... Oct 2012Advances in simulation techniques and computing hardware have created a substantial overlap between the timescales accessible to atomic-level simulations and those on...
Advances in simulation techniques and computing hardware have created a substantial overlap between the timescales accessible to atomic-level simulations and those on which the fastest-folding proteins fold. Here we demonstrate, using simulations of four variants of the human villin headpiece, how simulations of spontaneous folding and unfolding can provide direct access to thermodynamic and kinetic quantities such as folding rates, free energies, folding enthalpies, heat capacities, Φ-values, and temperature-jump relaxation profiles. The quantitative comparison of simulation results with various forms of experimental data probing different aspects of the folding process can facilitate robust assessment of the accuracy of the calculations while providing a detailed structural interpretation for the experimental observations. In the example studied here, the analysis of folding rates, Φ-values, and folding pathways provides support for the notion that a norleucine double mutant of villin folds five times faster than the wild-type sequence, but following a slightly different pathway. This work showcases how computer simulation has now developed into a mature tool for the quantitative computational study of protein folding and dynamics that can provide a valuable complement to experimental techniques.
Topics: Computer Simulation; Kinetics; Models, Molecular; Protein Folding; Thermodynamics
PubMed: 22822217
DOI: 10.1073/pnas.1201811109 -
International Journal of Molecular... Apr 2009Kinetic studies of the early events in cytochrome c folding are reviewed with a focus on the evidence for folding intermediates on the submillisecond timescale. Evidence... (Review)
Review
Kinetic studies of the early events in cytochrome c folding are reviewed with a focus on the evidence for folding intermediates on the submillisecond timescale. Evidence from time-resolved absorption, circular dichroism, magnetic circular dichroism, fluorescence energy and electron transfer, small-angle X-ray scattering and amide hydrogen exchange studies on the t < or = 1 ms timescale reveals a picture of cytochrome c folding that starts with the approximately 1-micros conformational diffusion dynamics of the unfolded chains. A fractional population of the unfolded chains collapses on the 1 - 100 micros timescale to a compact intermediate I(C) containing some native-like secondary structure. Although the existence and nature of I(C) as a discrete folding intermediate remains controversial, there is extensive high time-resolution kinetic evidence for the rapid formation of I(C) as a true intermediate, i.e., a metastable state separated from the unfolded state by a discrete free energy barrier. Final folding to the native state takes place on millisecond and longer timescales, depending on the presence of kinetic traps such as heme misligation and proline mis-isomerization. The high folding rates observed in equilibrium molten globule models suggest that I(C) may be a productive folding intermediate. Whether it is an obligatory step on the pathway to the high free energy barrier associated with millisecond timescale folding to the native state, however, remains to be determined.
Topics: Circular Dichroism; Cytochromes c; Kinetics; Protein Folding; Protein Structure, Secondary; Protein Unfolding; Thermodynamics; Time Factors
PubMed: 19468320
DOI: 10.3390/ijms10041476 -
Proceedings of the National Academy of... Jun 2000We use a free energy functional theory to elucidate general properties of heterogeneously ordering, fast folding proteins, and we test our conclusions with lattice...
We use a free energy functional theory to elucidate general properties of heterogeneously ordering, fast folding proteins, and we test our conclusions with lattice simulations. We find that both structural and energetic heterogeneity can lower the free energy barrier to folding. Correlating stronger contact energies with entropically likely contacts of a given native structure lowers the barrier, and anticorrelating the energies has the reverse effect. Designing in relatively mild energetic heterogeneity can eliminate the barrier completely at the transition temperature. Sequences with native energies tuned to fold uniformly, as well as sequences tuned to fold reliably by a single or a few routes, are rare. Sequences with weak native energetic heterogeneity are more common; their folding kinetics is more strongly determined by properties of the native structure. Sequences with different distributions of stability throughout the protein may still be good folders to the same structure. A measure of folding route narrowness is introduced that correlates with rate and that can give information about the intrinsic biases in ordering arising from native topology. This theoretical framework allows us to investigate systematically the coupled effects of energy and topology in protein folding and to interpret recent experiments that investigate these effects.
Topics: Protein Folding; Thermodynamics
PubMed: 10841554
DOI: 10.1073/pnas.97.12.6509 -
International Journal of Molecular... Mar 2022The folding of lysozyme in glycerol was monitored by the fast scanning calorimetry technique. Application of a temperature-time profile with an isothermal segment for...
The folding of lysozyme in glycerol was monitored by the fast scanning calorimetry technique. Application of a temperature-time profile with an isothermal segment for refolding allowed assessment of the state of the non-equilibrium protein ensemble and gave information on the kinetics of folding. We found that the non-equilibrium protein ensemble mainly contains a mixture of unfolded and folded protein forms and partially folded intermediates, and enthalpic barriers control the kinetics of the process. Lysozyme folding in glycerol follows the same or similar triangular mechanism described in the literature for folding in water. The unfolding enthalpy of the intermediate must be no lower than 70% of the folded form, while the activation barrier for the unfolding of the intermediate (ca. 140 kJ/mol) is about 100 kJ/mol lower than that of the folded form (ca. 240-260 kJ/mol).
Topics: Calorimetry; Glycerol; Hydrogen-Ion Concentration; Kinetics; Muramidase; Protein Denaturation; Protein Folding; Proteins; Thermodynamics
PubMed: 35269914
DOI: 10.3390/ijms23052773 -
Protein Science : a Publication of the... Apr 1995General principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models... (Review)
Review
General principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models represent protein chains at a rudimentary level, but they involve few parameters, approximations, or implicit biases, and they allow complete explorations of conformational and sequence spaces. Such simulations have resulted in testable predictions that are sometimes unanticipated: The folding code is mainly binary and delocalized throughout the amino acid sequence. The secondary and tertiary structures of a protein are specified mainly by the sequence of polar and nonpolar monomers. More specific interactions may refine the structure, rather than dominate the folding code. Simple exact models can account for the properties that characterize protein folding: two-state cooperativity, secondary and tertiary structures, and multistage folding kinetics--fast hydrophobic collapse followed by slower annealing. These studies suggest the possibility of creating "foldable" chain molecules other than proteins. The encoding of a unique compact chain conformation may not require amino acids; it may require only the ability to synthesize specific monomer sequences in which at least one monomer type is solvent-averse.
Topics: Amino Acid Sequence; Biological Evolution; Hydrogen Bonding; Models, Molecular; Molecular Sequence Data; Mutation; Protein Conformation; Protein Denaturation; Protein Folding; Temperature; Thermodynamics
PubMed: 7613459
DOI: 10.1002/pro.5560040401 -
Protein Science : a Publication of the... Jun 1996Future research on protein folding must confront two serious dilemmas. (1) It may never be possible to observe at high resolution the very important structures that form... (Review)
Review
Future research on protein folding must confront two serious dilemmas. (1) It may never be possible to observe at high resolution the very important structures that form in the first few milliseconds of the refolding reaction. (2) The energy functions used to predict structure from sequence will always be approximations of the true energy function. One strategy to resolve both dilemmas is to view protein folding from a different perspective, one that no longer emphasizes time and unique trajectories through conformation space. Instead, free energy replaces time as the reaction coordinate, and ensembles of equilibrium states of partially folded proteins are analyzed in place of trajectories of one protein chain through conformation space, either in vitro or in silico. Initial characterization of the folding of staphylococcal nuclease within this alternative conceptual framework has led to an equilibrium folding pathway with several surprising features. In addition to the finding of two bundles of four hydrophobic segments containing both native and non-native interactions, a gradient in relative stability of different substructures has been identified, with the most stable interactions located toward the amino terminus and the least stable toward the carboxy terminus. Hydrophobic bundles with up-down topology and stability gradients may be two examples of numerous tactics used by proteins to facilitate rapid folding and minimize aggregation. As NMR methods for structural analysis of partially folded proteins are refined, higher resolution descriptions of the structure and dynamics of the polypeptide chain outside the native state may provide many insights into the processes and energetics underlying the self-assembly of folded structure.
Topics: Forecasting; Glycerol; Magnetic Resonance Spectroscopy; Micrococcal Nuclease; Protein Conformation; Protein Denaturation; Protein Folding; Proteins; Surface Properties; Urea
PubMed: 8762131
DOI: 10.1002/pro.5560050602 -
Protein Science : a Publication of the... Apr 2022Over the past quarter century, my engagement with the protein society has allowed me to witness first-hand the evolution of our deepening understanding of the complexity...
Over the past quarter century, my engagement with the protein society has allowed me to witness first-hand the evolution of our deepening understanding of the complexity of protein folding landscapes. During my own evolution as a protein scientist, my passion for protein folding has deepened into an obsession with mapping and decoding the thermodynamic and kinetic secrets of protein landscapes-especially those of rebel proteins, whose "nontraditional" behavior has challenged our paradigms and inspired the expansion of our models and methods. It is perhaps not surprising that I see parallels in the evolution of the landscape framework and in the development of our own trajectories as humans in Science, Technology, Engineering and Mathematics (STEM). Just as with proteins, however, we need to recognize that our individual human landscapes are not isolated from our local departmental and institutional communities, and are integrated into the larger networks of our STEM disciplines, academia, industry, and/or government, not to mention society. My experience with hundreds of participants in the Being Human in STEM (HSTEM) initiative that Amherst College undergraduates and I co-founded in 2016 has helped me find hope for STEM and humanity. If we commit to reconciling our identities as scientists with our responsibilities as human beings, together we can accelerate the evolution of individual, community, and societal landscapes to contribute to addressing the dire challenges facing our planet.
Topics: Humans; Mathematics; Protein Folding; Proteins
PubMed: 35048424
DOI: 10.1002/pro.4278