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Biopolymers Nov 2013The recent revolution in optics and instrumentation has enabled the study of protein folding using extremely low mechanical forces as the denaturant. This exciting...
The recent revolution in optics and instrumentation has enabled the study of protein folding using extremely low mechanical forces as the denaturant. This exciting development has led to the observation of the protein folding process at single molecule resolution and its response to mechanical force. Here, we describe the principles and experimental details of force spectroscopy on proteins, with a focus on the optical tweezers instrument. Several recent results will be discussed to highlight the importance of this technique in addressing a variety of questions in the protein folding field.
Topics: Microscopy, Atomic Force; Optical Tweezers; Protein Folding; Proteins; Spectrum Analysis
PubMed: 23784721
DOI: 10.1002/bip.22321 -
Journal of Chemical Theory and... Mar 2024Protein folding is a fascinating, not fully understood phenomenon in biology. Molecular dynamics (MD) simulations are an invaluable tool to study conformational changes...
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.
Topics: Water; Protein Folding; Proteins; Molecular Dynamics Simulation; Molecular Conformation; Thermodynamics; Protein Conformation; Protein Unfolding
PubMed: 38373307
DOI: 10.1021/acs.jctc.3c01106 -
International Review of Cell and... 2013This review focuses on the current view of the interaction between the β-barrel scaffold of fluorescent proteins and their unique chromophore located in the internal... (Review)
Review
This review focuses on the current view of the interaction between the β-barrel scaffold of fluorescent proteins and their unique chromophore located in the internal helix. The chromophore originates from the polypeptide chain and its properties are influenced by the surrounding protein matrix of the β-barrel. On the other hand, it appears that a chromophore tightens the β-barrel scaffold and plays a crucial role in its stability. Furthermore, the presence of a mature chromophore causes hysteresis of protein unfolding and refolding. We survey studies measuring protein unfolding and refolding using traditional methods as well as new approaches, such as mechanical unfolding and reassembly of truncated fluorescent proteins. We also analyze models of fluorescent protein unfolding and refolding obtained through different approaches, and compare the results of protein folding in vitro to co-translational folding of a newly synthesized polypeptide chain.
Topics: Luminescent Proteins; Models, Molecular; Protein Refolding; Protein Stability; Protein Structure, Secondary; Protein Unfolding
PubMed: 23351712
DOI: 10.1016/B978-0-12-407699-0.00004-2 -
Biochimica Et Biophysica Acta Apr 2012Transmembrane transporters are responsible for maintaining a correct internal cellular environment. The inherent flexibility of transporters together with their... (Review)
Review
Transmembrane transporters are responsible for maintaining a correct internal cellular environment. The inherent flexibility of transporters together with their hydrophobic environment means that they are challenging to study in vitro, but recently significant progress been made. This review will focus on in vitro stability and folding studies of transmembrane alpha helical transporters, including reversible folding systems and thermal denaturation. The successful re-assembly of a small number of ATP binding cassette transporters is also described as this is a significant step forward in terms of understanding the folding and assembly of these more complex, multi-subunit proteins. The studies on transporters discussed here represent substantial advances for membrane protein studies as well as for research into protein folding. The work demonstrates that large flexible hydrophobic proteins are within reach of in vitro folding studies, thus holding promise for furthering knowledge on the structure, function and biogenesis of ubiquitous membrane transporter families. This article is part of a Special Issue entitled: Protein Folding in Membranes.
Topics: Membrane Transport Proteins; Models, Biological; Protein Denaturation; Protein Folding; Protein Refolding; Protein Stability
PubMed: 22100867
DOI: 10.1016/j.bbamem.2011.11.006 -
Archives of Biochemistry and Biophysics Jan 2008Protein aggregation has now become recognised as an important and generic aspect of protein energy landscapes. Since the discovery that numerous human diseases are... (Review)
Review
Protein aggregation has now become recognised as an important and generic aspect of protein energy landscapes. Since the discovery that numerous human diseases are caused by protein aggregation, the biophysical characterisation of misfolded states and their aggregation mechanisms has received increased attention. Utilising experimental techniques and computational approaches established for the analysis of protein folding reactions has ensured rapid advances in the study of pathways leading to amyloid fibrils and amyloid-related aggregates. Here we describe recent experimental and theoretical advances in the elucidation of the conformational properties of dynamic, heterogeneous and/or insoluble protein ensembles populated on complex, multidimensional protein energy landscapes. We discuss current understanding of aggregation mechanisms in this context and describe how the synergy between biochemical, biophysical and cell-biological experiments are beginning to provide detailed insights into the partitioning of non-native species between protein folding and aggregation pathways.
Topics: Amyloid; Protein Conformation; Protein Folding
PubMed: 17588526
DOI: 10.1016/j.abb.2007.05.015 -
IUBMB Life Jun 2009Protein research is generally recognized as experimental science and knowledge of protein science is not constructed axiomatically. In this article, we show that much of... (Review)
Review
Protein research is generally recognized as experimental science and knowledge of protein science is not constructed axiomatically. In this article, we show that much of our present knowledge of protein science is explainable by principles of protein thermodynamic structure theory. A deductive system for protein knowledge has been developed and several fundamental questions of protein science can be theoretically resolved.
Topics: Protein Conformation; Protein Denaturation; Protein Folding; Protein Stability; Proteins; Receptors, Cell Surface; Receptors, Drug; Thermodynamics
PubMed: 19472177
DOI: 10.1002/iub.214 -
Molecules (Basel, Switzerland) Nov 2020High-hydrostatic pressure is an alternative perturbation method that can be used to destabilize globular proteins. Generally perfectly reversible, pressure exerts local... (Review)
Review
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.
Topics: Hydrostatic Pressure; Kinetics; Magnetic Resonance Spectroscopy; Models, Molecular; Models, Theoretical; Pressure; Protein Conformation; Protein Folding; Protein Unfolding; Proteins; Structure-Activity Relationship; Thermodynamics
PubMed: 33256081
DOI: 10.3390/molecules25235551 -
Proceedings of the National Academy of... Nov 2006Under physiological conditions, a protein undergoes a spontaneous disorder order transition called "folding." The protein polymer is highly flexible when unfolded but... (Review)
Review
Under physiological conditions, a protein undergoes a spontaneous disorder order transition called "folding." The protein polymer is highly flexible when unfolded but adopts its unique native, three-dimensional structure when folded. Current experimental knowledge comes primarily from thermodynamic measurements in solution or the structures of individual molecules, elucidated by either x-ray crystallography or NMR spectroscopy. From the former, we know the enthalpy, entropy, and free energy differences between the folded and unfolded forms of hundreds of proteins under a variety of solvent/cosolvent conditions. From the latter, we know the structures of approximately 35,000 proteins, which are built on scaffolds of hydrogen-bonded structural elements, alpha-helix and beta-sheet. Anfinsen showed that the amino acid sequence alone is sufficient to determine a protein's structure, but the molecular mechanism responsible for self-assembly remains an open question, probably the most fundamental open question in biochemistry. This perspective is a hybrid: partly review, partly proposal. First, we summarize key ideas regarding protein folding developed over the past half-century and culminating in the current mindset. In this view, the energetics of side-chain interactions dominate the folding process, driving the chain to self-organize under folding conditions. Next, having taken stock, we propose an alternative model that inverts the prevailing side-chain/backbone paradigm. Here, the energetics of backbone hydrogen bonds dominate the folding process, with preorganization in the unfolded state. Then, under folding conditions, the resultant fold is selected from a limited repertoire of structural possibilities, each corresponding to a distinct hydrogen-bonded arrangement of alpha-helices and/or strands of beta-sheet.
Topics: Animals; Humans; Hydrogen Bonding; Models, Molecular; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Thermodynamics
PubMed: 17075053
DOI: 10.1073/pnas.0606843103 -
Biophysical Journal Aug 2021The folding stability of a protein is governed by the free-energy difference between its folded and unfolded states, which results from a delicate balance of much larger...
The folding stability of a protein is governed by the free-energy difference between its folded and unfolded states, which results from a delicate balance of much larger but almost compensating enthalpic and entropic contributions. The balance can therefore easily be shifted by an external disturbance, such as a mutation of a single amino acid or a change of temperature, in which case the protein unfolds. Effects such as cold denaturation, in which a protein unfolds because of cooling, provide evidence that proteins are strongly stabilized by the solvent entropy contribution to the free-energy balance. However, the molecular mechanisms behind this solvent-driven stability, their quantitative contribution in relation to other free-energy contributions, and how the involved solvent thermodynamics is affected by individual amino acids are largely unclear. Therefore, we addressed these questions using atomistic molecular dynamics simulations of the small protein Crambin in its native fold and a molten-globule-like conformation, which here served as a model for the unfolded state. The free-energy difference between these conformations was decomposed into enthalpic and entropic contributions from the protein and spatially resolved solvent contributions using the nonparametric method Per|Mut. From the spatial resolution, we quantified the local effects on the solvent free-energy difference at each amino acid and identified dependencies of the local enthalpy and entropy on the protein curvature. We identified a strong stabilization of the native fold by almost 500 kJ mol due to the solvent entropy, revealing it as an essential contribution to the total free-energy difference of (53 ± 84) kJ mol. Remarkably, more than half of the solvent entropy contribution arose from induced water correlations.
Topics: Entropy; Plant Proteins; Protein Conformation; Protein Denaturation; Protein Folding; Thermodynamics
PubMed: 34087209
DOI: 10.1016/j.bpj.2021.05.019 -
Proceedings of the National Academy of... Sep 2018The presence of conflicting interactions, or frustration, determines how fast biomolecules can explore their configurational landscapes. Recent experiments have provided...
The presence of conflicting interactions, or frustration, determines how fast biomolecules can explore their configurational landscapes. Recent experiments have provided cases of systems with slow reconfiguration dynamics, perhaps arising from frustration. While it is well known that protein folding speed and mechanism are strongly affected by the protein native structure, it is still unknown how the response to frustration is modulated by the protein topology. We explore the effects of nonnative interactions in the reconfigurational and folding dynamics of proteins with different sizes and topologies. We find that structural correlations related to the folded state size and topology play an important role in determining the folding kinetics of proteins that otherwise have the same amount of nonnative interactions. In particular, we find that the reconfiguration dynamics of α-helical proteins are more susceptible to frustration than β-sheet proteins of the same size. Our results may explain recent experimental findings and suggest that attempts to measure the degree of frustration due to nonnative interactions might be more successful with α-helical proteins.
Topics: Models, Chemical; Protein Folding; Protein Structure, Secondary; Proteins
PubMed: 30150375
DOI: 10.1073/pnas.1801406115