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Nature Nanotechnology Apr 2013Cells are divided into compartments and separated from the environment by lipid bilayer membranes. Essential molecules are transported back and forth across the...
Cells are divided into compartments and separated from the environment by lipid bilayer membranes. Essential molecules are transported back and forth across the membranes. We have investigated how folded proteins use narrow transmembrane pores to move between compartments. During this process, the proteins must unfold. To examine co-translocational unfolding of individual molecules, we tagged protein substrates with oligonucleotides to enable potential-driven unidirectional movement through a model protein nanopore, a process that differs fundamentally from extension during force spectroscopy measurements. Our findings support a four-step translocation mechanism for model thioredoxin substrates. First, the DNA tag is captured by the pore. Second, the oligonucleotide is pulled through the pore, causing local unfolding of the C terminus of the thioredoxin adjacent to the pore entrance. Third, the remainder of the protein unfolds spontaneously. Finally, the unfolded polypeptide diffuses through the pore into the recipient compartment. The unfolding pathway elucidated here differs from those revealed by denaturation experiments in solution, for which two-state mechanisms have been proposed.
Topics: Animals; Bacterial Toxins; Electricity; Escherichia coli; Hemolysin Proteins; Kinetics; Lipid Bilayers; Models, Molecular; Mutation; Nanopores; Oligonucleotides; Protein Unfolding; Rabbits; Thioredoxins; Urea
PubMed: 23474543
DOI: 10.1038/nnano.2013.22 -
Nanoscale Jun 2016An assortment of biological processes, like protein degradation and the transport of proteins across membranes, depend on protein unfolding events mediated by nanopore...
An assortment of biological processes, like protein degradation and the transport of proteins across membranes, depend on protein unfolding events mediated by nanopore interfaces. In this work, we exploit fully atomistic simulations of an artificial, CNT-based nanopore to investigate the nature of ubiquitin unfolding. With one end of the protein subjected to an external force, we observe non-canonical unfolding behaviour as ubiquitin is pulled through the pore opening. Secondary structural elements are sequentially detached from the protein and threaded into the nanotube, interestingly, the remaining part maintains native-like characteristics. The constraints of the nanopore interface thus facilitate the formation of stable "unfoldon" motifs above the nanotube aperture that can exist in the absence of specific native contacts with the other secondary structure. Destruction of these unfoldons gives rise to distinct force peaks in our simulations, providing us with a sensitive probe for studying the kinetics of serial unfolding events. Our detailed analysis of nanopore-mediated protein unfolding events not only provides insight into how related processes might proceed in the cell, but also serves to deepen our understanding of structural arrangements which form the basis for protein conformational stability.
Topics: Models, Molecular; Nanopores; Nanotubes, Carbon; Protein Conformation; Protein Folding; Protein Unfolding; Proteins
PubMed: 26899409
DOI: 10.1039/c6nr00410e -
Protein Science : a Publication of the... Dec 2022Protein folding and unfolding is a complex process, underscored by the many proteotoxic diseases associated with misfolded proteins. Mapping pathways from a native...
Protein folding and unfolding is a complex process, underscored by the many proteotoxic diseases associated with misfolded proteins. Mapping pathways from a native structure to an unfolded protein or vice versa, identifying the intermediates, and defining the role of sequence and structure en route remain outstanding problems in the field. It is even more challenging to capture the events at atomistic resolution. X-ray diffraction has so far been used to understand how urea interacts with and unfolds two stable globular proteins. Here, we present the case study on PSMD10 , a prototype for a moderately stable, non-globular repeat protein, long and rigid, with its termini located at either end. We define structural changes in the time dimension using low urea concentrations to arrive at the following conclusions. (a) Unfolding is rapidly initiated at the C-terminus, slowly at the N-terminus, and proceeds inwards from both ends. (b) C-terminus undergoes an α to 3 helix transition, representing the structure of a potential early unfolding intermediate before disorder sets in. (c) Distinct and progressive changes in the electrostatic landscape of PSMD10 were observed, indicative of conformational changes in the seemingly inflexible motif involved in protein-protein interaction. We believe this unique study will open up the field for better and bolder queries and increase the choice of model proteins for a better understanding of the challenging problems of protein folding, protein interactions, protein degradation, and diseases associated with misfolding.
Topics: Protein Denaturation; Urea; Ankyrin Repeat; Protein Folding; X-Ray Diffraction; Protein Conformation; Protein Unfolding
PubMed: 36382986
DOI: 10.1002/pro.4515 -
Biophysical Journal Mar 2018The folding dynamics of proteins at the single-molecule level has been studied with single-molecule force spectroscopy experiments for 20 years, but a common...
The folding dynamics of proteins at the single-molecule level has been studied with single-molecule force spectroscopy experiments for 20 years, but a common standardized method for the analysis of the collected data and for sharing among the scientific community members is still not available. We have developed a new open-source tool-Fodis-for the analysis of the force-distance curves obtained in single-molecule force spectroscopy experiments, providing almost automatic processing, analysis, and classification of the obtained data. Our method provides also a classification of the possible unfolding pathways and the structural heterogeneity present during the unfolding of proteins.
Topics: Biomechanical Phenomena; Microscopy, Atomic Force; Protein Unfolding; Software
PubMed: 29590583
DOI: 10.1016/j.bpj.2018.02.004 -
Molecules (Basel, Switzerland) Jul 2021Protein folding is important for protein homeostasis/proteostasis in the human body. We have established the ability to manipulate protein unfolding/refolding for...
Protein folding is important for protein homeostasis/proteostasis in the human body. We have established the ability to manipulate protein unfolding/refolding for β-lactoglobulin using the induced mechanical energy in the thin film microfluidic vortex fluidic device (VFD) with monitoring as such using an aggregation-induced emission luminogen (AIEgen), TPE-MI. When denaturant (guanidine hydrochloride) is present with β-lactoglobulin, the VFD accelerates the denaturation reaction in a controlled way. Conversely, rapid renaturation of the unfolded protein occurs in the VFD in the absence of the denaturant. The novel TPE-MI reacts with exposed cysteine thiol when the protein unfolds, as established with an increase in fluorescence intensity. TPE-MI provides an easy and accurate way to monitor the protein folding, with comparable results established using conventional circular dichroism. The controlled VFD-mediated protein folding coupled with in situ bioprobe AIEgen monitoring is a viable methodology for studying the denaturing of proteins.
Topics: Biosensing Techniques; Circular Dichroism; Cysteine; Guanidine; Humans; Kinetics; Lactoglobulins; Protein Conformation; Protein Denaturation; Protein Folding; Protein Refolding; Protein Unfolding; Proteins; Proteostasis; Spectrometry, Fluorescence
PubMed: 34299548
DOI: 10.3390/molecules26144273 -
Advances in Protein Chemistry 2001Although much has been learned about the structure and function of Clp chaperones and their role in proteolysis, the mechanism of protein unfolding catalyzed by Clp... (Review)
Review
Although much has been learned about the structure and function of Clp chaperones and their role in proteolysis, the mechanism of protein unfolding catalyzed by Clp ATPases and the mechanism of translocation of the unfolded proteins from Clp ATPases to partner proteases remain unsolved puzzles. However, models in which mechanical force is used to destabilize the structure of the substrate in a processive and directional manner are probable. It also seems likely that when ClpA ATPases are associated with proteases, unfolding is coupled to extrusion of the unfolded protein into the proteolytic cavity. In summary, it is anticipated that the large family of Clp ATPases will accomplish their many important cellular functions by similar mechanisms and what has been learned by studying the prokaryotic members reviewed here will shed a great deal of light on all members of the family.
Topics: Adenosine Triphosphatases; Hydrolysis; Molecular Chaperones; Protein Denaturation; Substrate Specificity
PubMed: 11868279
DOI: 10.1016/s0065-3233(01)59013-0 -
Analytical and Bioanalytical Chemistry Feb 2023Electrospray ionization mass spectrometry (ESI-MS) experiments, including ion mobility spectrometry mass spectrometry (ESI-IMS-MS) and electron capture dissociation...
Electrospray ionization mass spectrometry (ESI-MS) experiments, including ion mobility spectrometry mass spectrometry (ESI-IMS-MS) and electron capture dissociation (ECD) of proteins ionized from aqueous solutions, have been used for the study of solution-like structures of intact proteins. By mixing aqueous proteins with denaturants online before ESI, the amount of protein unfolding can be precisely controlled and rapidly analyzed, permitting the characterization of protein folding intermediates in protein folding pathways. Herein, we mixed various pH solutions online with aqueous cytochrome C for unfolding and characterizing its unfolding intermediates with ESI-MS charge state distribution measurements, IMS, and ECD. The presence of folding intermediates and unfolded cytochrome c structures were detected from changes in charge states, arrival time distributions (ATDs), and ECD. We also compared structures from nondenaturing and denaturing solution mixtures measured under "gentle" (i.e., low energy) ion transmission conditions with structures measured under "harsh" (i.e., higher energy) transmission. This work confirms that when using "gentle" instrument conditions, the gas-phase cytochrome c ions reflect attributes of the various solution-phase structures. However, "harsh" conditions that maximize ion transmission produce extended structures that no longer correlate with changes in solution structure.
Topics: Cytochromes c; Ion Mobility Spectrometry; Electrons; Proteins; Spectrometry, Mass, Electrospray Ionization; Protein Unfolding; Acids; Ions; Water
PubMed: 36622393
DOI: 10.1007/s00216-022-04501-w -
Scientific Reports Nov 2017Studies of protein unfolding mechanisms are critical for understanding protein functions inside cells, de novo protein design as well as defining the role of protein...
Studies of protein unfolding mechanisms are critical for understanding protein functions inside cells, de novo protein design as well as defining the role of protein misfolding in neurodegenerative disorders. Calorimetry has proven indispensable in this regard for recording full energetic profiles of protein unfolding and permitting data fitting based on unfolding pathway models. While both kinetic and thermodynamic protein stability are analysed by varying scan rates and reheating, the latter is rarely used in curve-fitting, leading to a significant loss of information from experiments. To extract this information, we propose fitting both first and second scans simultaneously. Four most common single-peak transition models are considered: (i) fully reversible, (ii) fully irreversible, (iii) partially reversible transitions, and (iv) general three-state models. The method is validated using calorimetry data for chicken egg lysozyme, mutated Protein A, three wild-types of haloalkane dehalogenases, and a mutant stabilized by protein engineering. We show that modelling of reheating increases the precision of determination of unfolding mechanisms, free energies, temperatures, and heat capacity differences. Moreover, this modelling indicates whether alternative refolding pathways might occur upon cooling. The Matlab-based data fitting software tool and its user guide are provided as a supplement.
Topics: Animals; Calorimetry; Chick Embryo; Kinetics; Muramidase; Protein Engineering; Protein Unfolding
PubMed: 29176711
DOI: 10.1038/s41598-017-16360-y -
International Journal of Molecular... Mar 2021When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be...
When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-β or mixed-α/β protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway.
Topics: Adaptor Proteins, Signal Transducing; Humans; Kinetics; Magnetic Resonance Spectroscopy; Models, Molecular; Protein Conformation; Protein Conformation, alpha-Helical; Protein Denaturation; Protein Domains; Protein Unfolding; Temperature; Thermodynamics
PubMed: 33808390
DOI: 10.3390/ijms22073597 -
Protein Engineering, Design & Selection... Feb 2021We review the background, theory and general equations for the analysis of equilibrium protein unfolding experiments, focusing on denaturant and heat-induced unfolding.... (Review)
Review
We review the background, theory and general equations for the analysis of equilibrium protein unfolding experiments, focusing on denaturant and heat-induced unfolding. The primary focus is on the thermodynamics of reversible folding/unfolding transitions and the experimental methods that are available for extracting thermodynamic parameters. We highlight the importance of modelling both how the folding equilibrium depends on a perturbing variable such as temperature or denaturant concentration, and the importance of modelling the baselines in the experimental observables.
Topics: Kinetics; Protein Denaturation; Protein Folding; Protein Stability; Temperature; Thermodynamics
PubMed: 33724431
DOI: 10.1093/protein/gzab002