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Chembiochem : a European Journal of... Aug 2023This review aims to analyse the role of solution nuclear magnetic resonance spectroscopy in pressure-induced in vitro studies of protein unfolding. Although this... (Review)
Review
This review aims to analyse the role of solution nuclear magnetic resonance spectroscopy in pressure-induced in vitro studies of protein unfolding. Although this transition has been neglected for many years because of technical difficulties, it provides important information about the forces that keep protein structure together. We first analyse what pressure unfolding is, then provide a critical overview of how NMR spectroscopy has contributed to the field and evaluate the observables used in these studies. Finally, we discuss the commonalities and differences between pressure-, cold- and heat-induced unfolding. We conclude that, despite specific peculiarities, in both cold and pressure denaturation the important contribution of the state of hydration of nonpolar side chains is a major factor that determines the pressure dependence of the conformational stability of proteins.
Topics: Protein Denaturation; Proteins; Magnetic Resonance Spectroscopy; Protein Unfolding; Protein Conformation; Thermodynamics; Protein Folding; Cold Temperature
PubMed: 37154795
DOI: 10.1002/cbic.202300164 -
Organic & Biomolecular Chemistry Nov 2018Molecular photoswitches are a class of chemical structures that can readily isomerize between distinct geometries upon irradiation with light. Molecular photoswitches... (Review)
Review
Molecular photoswitches are a class of chemical structures that can readily isomerize between distinct geometries upon irradiation with light. Molecular photoswitches are utilized to control protein structure and function with temporal and spatial precision. In this review, we summarize the recent progress in the development of azobenzene-based molecular photoswitches and their applications in the photocontrol of protein structure and function. For clarity of discussion, we divide the known photoswitchable proteins into different categories: protein motifs, ion channels, receptors, and enzymes. Basic approaches and considerations for the structure-guided design of photoswitchable ligands are discussed. The applications and limitations of current photoswitches are also discussed.
Topics: Azo Compounds; Humans; Light; Protein Conformation; Protein Folding; Proteins
PubMed: 30375620
DOI: 10.1039/c8ob02157k -
Current Medicinal Chemistry 2022
Topics: Humans; Protein Conformation; Protein Folding
PubMed: 35532256
DOI: 10.2174/0929867329666220508173020 -
Emerging Topics in Life Sciences May 2021Protein folding is crucial for normal physiology including development and healthy aging, and failure of this process is related to the pathology of diseases including...
Protein folding is crucial for normal physiology including development and healthy aging, and failure of this process is related to the pathology of diseases including neurodegeneration and cancer. Early thermodynamic and kinetic studies based on the unfolding and refolding equilibrium of individual proteins in the test tube have provided insight into the fundamental principles of protein folding, although the problem of predicting how any given protein will fold remains unsolved. Protein folding within cells is a more complex issue than folding of purified protein in isolation, due to the complex interactions within the cellular environment, including post-translational modifications of proteins, the presence of macromolecular crowding in cells, and variations in the cellular environment, for example in cancer versus normal cells. Development of biophysical approaches including fluorescence resonance energy transfer (FRET) and nuclear magnetic resonance (NMR) techniques and cellular manipulations including microinjection and insertion of noncanonical amino acids has allowed the study of protein folding in living cells. Furthermore, biophysical techniques such as single-molecule fluorescence spectroscopy and optical tweezers allows studies of simplified systems at the single molecular level. Combining in-cell techniques with the powerful detail that can be achieved from single-molecule studies allows the effects of different cellular components including molecular chaperones to be monitored, providing us with comprehensive understanding of the protein folding process. The application of biophysical techniques to the study of protein folding is arming us with knowledge that is fundamental to the battle against cancer and other diseases related to protein conformation or protein-protein interactions.
Topics: Kinetics; Molecular Chaperones; Protein Conformation; Protein Folding; Thermodynamics
PubMed: 33660767
DOI: 10.1042/ETLS20200317 -
F1000Research 2021Proteins fold robustly and reproducibly , but many cannot fold in isolation from cellular components. Despite the remarkable progress that has been achieved by the...
Proteins fold robustly and reproducibly , but many cannot fold in isolation from cellular components. Despite the remarkable progress that has been achieved by the artificial intelligence approaches in predicting the protein native conformations, the pathways that lead to such conformations, either or , remain largely unknown. The slow progress in recapitulating protein folding pathways may be an indication of the fundamental deficiencies in our understanding of folding as it occurs in nature. Here we consider the possibility that protein folding in living cells may not be driven solely by the decrease in Gibbs free energy and propose that protein folding should be modeled as an active energy-dependent process. The mechanism of action of such a protein folding machine might include direct manipulation of the peptide backbone. To show the feasibility of a protein folding machine, we conducted molecular dynamics simulations that were augmented by the application of mechanical force to rotate the C-terminal amino acid while simultaneously limiting the N-terminal amino acid movements. Remarkably, the addition of this simple manipulation of peptide backbones to the standard molecular dynamics simulation indeed facilitated the formation of native structures in five diverse alpha-helical peptides. Steric clashes that arise in the peptides due to the forced directional rotation resulted in the behavior of the peptide backbone no longer resembling a freely jointed chain. These simulations show the feasibility of a protein folding machine operating under the conditions when the movements of the polypeptide backbone are restricted by applying external forces and constraints. Further investigation is needed to see whether such an effect may play a role during co-translational protein folding and how it can be utilized to facilitate folding of proteins in artificial environments.
Topics: Artificial Intelligence; Peptides; Protein Conformation; Protein Folding; Proteins
PubMed: 33633838
DOI: 10.12688/f1000research.28175.1 -
The Biochemical Journal Sep 2016Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a... (Review)
Review
Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats.
Topics: Protein Folding; Proteins; Thermodynamics
PubMed: 27574021
DOI: 10.1042/BCJ20160107 -
Physics of Life Reviews Jul 2017The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured folding times of... (Review)
Review
The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured folding times of single-domain globular proteins range from microseconds to hours: the difference (10-11 orders of magnitude) is the same as that between the life span of a mosquito and the age of the universe. This review describes physical theories of rates of overcoming the free-energy barrier separating the natively folded (N) and unfolded (U) states of protein chains in both directions: "U-to-N" and "N-to-U". In the theory of protein folding rates a special role is played by the point of thermodynamic (and kinetic) equilibrium between the native and unfolded state of the chain; here, the theory obtains the simplest form. Paradoxically, a theoretical estimate of the folding time is easier to get from consideration of protein unfolding (the "N-to-U" transition) rather than folding, because it is easier to outline a good unfolding pathway of any structure than a good folding pathway that leads to the stable fold, which is yet unknown to the folding protein chain. And since the rates of direct and reverse reactions are equal at the equilibrium point (as follows from the physical "detailed balance" principle), the estimated folding time can be derived from the estimated unfolding time. Theoretical analysis of the "N-to-U" transition outlines the range of protein folding rates in a good agreement with experiment. Theoretical analysis of folding (the "U-to-N" transition), performed at the level of formation and assembly of protein secondary structures, outlines the upper limit of protein folding times (i.e., of the time of search for the most stable fold). Both theories come to essentially the same results; this is not a surprise, because they describe overcoming one and the same free-energy barrier, although the way to the top of this barrier from the side of the unfolded state is very different from the way from the side of the native state; and both theories agree with experiment. In addition, they predict the maximal size of protein domains that fold under solely thermodynamic (rather than kinetic) control and explain the observed maximal size of the "foldable" protein domains.
Topics: Models, Molecular; Protein Folding; Proteins
PubMed: 28190683
DOI: 10.1016/j.plrev.2017.01.025 -
The Journal of Physical Chemistry. B Mar 2021One-third of the reported cases of light chain amyloidosis are related to the germ line λ6 family; remarkably, healthy individuals express this type of protein in just...
One-third of the reported cases of light chain amyloidosis are related to the germ line λ6 family; remarkably, healthy individuals express this type of protein in just 2% of the peripheral blood and bone marrow B-cells. The appearance of the disease has been related to the inherent properties of this protein family. A recombinant representative model for λ6 proteins called 6aJL2 containing the amino acid sequence encoded by the 6a and JL2 germ line genes was previously designed and synthesized to study the properties of this family. Previous work on 6aJL2 suggested a simple two-state folding model at 25 °C; no intermediate could be identified either by kinetics or by fluorescence and circular dichroism equilibrium studies, although the presence of an intermediate that is populated at ∼2.4 M urea was suggested by size exclusion chromatography. In this study we employed classic equilibrium and kinetic experiments and analysis to elucidate the detailed folding mechanism of this protein. We identify species that are kinetically accessible and/or are populated at equilibrium. We describe the presence of intermediate and native-like species and propose a five-species folding mechanism at 25 °C at short incubation times, similar to and consistent with those observed in other proteins of this fold. The formation of intermediates in the mechanism of 6aJL2 is faster than that proposed for a light chain, which could be an important distinction in the amyloidogenic potential of both germ lines.
Topics: Amino Acid Sequence; Amyloidosis; Circular Dichroism; Humans; Kinetics; Protein Denaturation; Protein Folding
PubMed: 33620231
DOI: 10.1021/acs.jpcb.0c08534 -
The Journal of Physical Chemistry. B Jul 2014Protein folding is a remarkably fast unimolecular reaction, spanning microseconds to hours at room temperature. Thus, free energy differences and activation barriers on... (Review)
Review
Protein folding is a remarkably fast unimolecular reaction, spanning microseconds to hours at room temperature. Thus, free energy differences and activation barriers on the free energy landscape of proteins are rather small. This opens up the possibility of living cells modulating their protein's landscapes, providing cells another way to control the function of their proteomes after transcriptional control, translational control, and post-translational modification. In this Feature Article, we discuss advances in physicochemical studies of protein stability and folding inside living cells. We focus in particular on our studies using fast relaxation imaging (FREI). Although the effect of the cell on protein free energy landscapes is only a few kT, the strong cooperativity of many folding and binding processes allows small modulation of the energy and entropy to produce a large population modulation. Lastly, we discuss some biomolecular processes that are particularly likely to be affected by in-cell modulation of the proteome, and thus of interest for quantitative physical chemistry studies.
Topics: Animals; Cells; Humans; Molecular Imaging; Protein Folding
PubMed: 24878167
DOI: 10.1021/jp501866v -
Antimicrobial Agents and Chemotherapy Apr 2017Antimicrobial peptides (AMPs) are expressed in various living organisms as first-line host defenses against potential harmful encounters in their surroundings. AMPs are... (Review)
Review
Antimicrobial peptides (AMPs) are expressed in various living organisms as first-line host defenses against potential harmful encounters in their surroundings. AMPs are short polycationic peptides exhibiting various antimicrobial activities. The principal antibacterial activity is attributed to the membrane-lytic mechanism which directly interferes with the integrity of the bacterial cell membrane and cell wall. In addition, a number of AMPs form a transmembrane channel in the membrane by self-aggregation or polymerization, leading to cytoplasm leakage and cell death. However, an increasing body of evidence has demonstrated that AMPs are able to exert intracellular inhibitory activities as the primary or supportive mechanisms to achieve efficient killing. In this review, we focus on the major intracellular targeting activities reported in AMPs, which include nucleic acids and protein biosynthesis and protein-folding, protease, cell division, cell wall biosynthesis, and lipopolysaccharide inhibition. These multifunctional AMPs could serve as the potential lead peptides for the future development of novel antibacterial agents with improved therapeutic profiles.
Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Cell Membrane; Cell Wall; Microbial Sensitivity Tests; Protein Biosynthesis; Protein Folding
PubMed: 28167546
DOI: 10.1128/AAC.02340-16