-
Biomolecules Sep 2020In this work, we investigate the role of folding/unfolding equilibrium in protein aggregation and formation of a gel network. Near the neutral pH and at a low buffer...
In this work, we investigate the role of folding/unfolding equilibrium in protein aggregation and formation of a gel network. Near the neutral pH and at a low buffer ionic strength, the formation of the gel network around unfolding conditions prevents investigations of protein aggregation. In this study, by deploying the fact that in lysozyme solutions the time of folding/unfolding is much shorter than the characteristic time of gelation, we have prevented gelation by rapidly heating the solution up to the unfolding temperature (~80 °C) for a short time (~30 min.) followed by fast cooling to the room temperature. Dynamic light scattering measurements show that if the gelation is prevented, nanosized irreversible aggregates (about 10-15 nm radius) form over a time scale of 10 days. These small aggregates persist and aggregate further into larger aggregates over several weeks. If gelation is not prevented, the nanosized aggregates become the building blocks for the gel network and define its mesh length scale. These results support our previously published conclusion on the nature of mesoscopic aggregates commonly observed in solutions of lysozyme, namely that aggregates do not form from lysozyme monomers in their native folded state. Only with the emergence of a small fraction of unfolded proteins molecules will the aggregates start to appear and grow.
Topics: Dynamic Light Scattering; Gels; Hot Temperature; Muramidase; Protein Aggregates; Protein Unfolding; Solutions
PubMed: 32887233
DOI: 10.3390/biom10091262 -
Trends in Biochemical Sciences Nov 2017Proteins are constantly challenged by environmental stress conditions that threaten their structure and function. Especially problematic are oxidative, acid, and severe... (Review)
Review
Proteins are constantly challenged by environmental stress conditions that threaten their structure and function. Especially problematic are oxidative, acid, and severe heat stress which induce very rapid and widespread protein unfolding and generate conditions that make canonical chaperones and/or transcriptional responses inadequate to protect the proteome. We review here recent advances in identifying and characterizing stress-activated chaperones which are inactive under non-stress conditions but become potent chaperones under specific protein-unfolding stress conditions. We discuss the post-translational mechanisms by which these chaperones sense stress, and consider the role that intrinsic disorder plays in their regulation and function. We examine their physiological roles under both non-stress and stress conditions, their integration into the cellular proteostasis network, and their potential as novel therapeutic targets.
Topics: Animals; Humans; Molecular Chaperones; Oxidative Stress; Protein Processing, Post-Translational; Protein Unfolding; Proteome
PubMed: 28893460
DOI: 10.1016/j.tibs.2017.08.006 -
International Journal of Molecular... Jul 2015Highly sophisticated mechanisms that modulate protein structure and function, which involve synthesis and degradation, have evolved to maintain cellular homeostasis.... (Review)
Review
Highly sophisticated mechanisms that modulate protein structure and function, which involve synthesis and degradation, have evolved to maintain cellular homeostasis. Perturbations in these mechanisms can lead to protein dysfunction as well as deleterious cell processes. Therefore in recent years the etiology of a great number of diseases has been attributed to failures in mechanisms that modulate protein structure. Interconnections among metabolic and cell signaling pathways are critical for homeostasis to converge on mechanisms associated with protein folding as well as for the preservation of the native structure of proteins. For instance, imbalances in secretory protein synthesis pathways lead to a condition known as endoplasmic reticulum (ER) stress which elicits the adaptive unfolded protein response (UPR). Therefore, taking this into consideration, a key part of this paper is developed around the protein folding phenomenon, and cellular mechanisms which support this pivotal condition. We provide an overview of chaperone protein function, UPR via, spatial compartmentalization of protein folding, proteasome role, autophagy, as well as the intertwining between these processes. Several diseases are known to have a molecular etiology in the malfunction of mechanisms responsible for protein folding and in the shielding of native structure, phenomena which ultimately lead to misfolded protein accumulation. This review centers on our current knowledge about pathways that modulate protein folding, and cell responses involved in protein homeostasis.
Topics: Animals; Homeostasis; Humans; Molecular Chaperones; Protein Unfolding; Unfolded Protein Response
PubMed: 26225966
DOI: 10.3390/ijms160817193 -
Proceedings of the National Academy of... Feb 2016Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments....
Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments. Unfolding rate, [Formula: see text], as a function of force f, obtained in single-molecule pulling experiments on src SH3 domain, exhibits upward curvature on a [Formula: see text] plot. Similar observations were reported for other proteins for the unfolding rate [Formula: see text]. These findings imply unfolding in these single-domain proteins involves a switch in the pathway as f or [Formula: see text] is increased from a low to a high value. We provide a unified theory demonstrating that if [Formula: see text] as a function of a perturbation (f or [Formula: see text]) exhibits upward curvature then the underlying energy landscape must be strongly multidimensional. Using molecular simulations we provide a structural basis for the switch in the pathways and dramatic shifts in the transition-state ensemble (TSE) in src SH3 domain as f is increased. We show that a single-point mutation shifts the upward curvature in [Formula: see text] to a lower force, thus establishing the malleability of the underlying folding landscape. Our theory, applicable to any perturbation that affects the free energy of the protein linearly, readily explains movement in the TSE in a β-sandwich (I27) protein and single-chain monellin as the denaturant concentration is varied. We predict that in the force range accessible in laser optical tweezer experiments there should be a switch in the unfolding pathways in I27 or its mutants.
Topics: Amino Acid Sequence; Animals; Biomechanical Phenomena; Chickens; Models, Molecular; Molecular Sequence Data; Movement; Mutant Proteins; Protein Unfolding; Thermodynamics; src Homology Domains
PubMed: 26818842
DOI: 10.1073/pnas.1515730113 -
Advanced Science (Weinheim,... Feb 2022Protein-based hydrogels have attracted great attention due to their excellent biocompatible properties, but often suffer from weak mechanical strength. Conventional...
Protein-based hydrogels have attracted great attention due to their excellent biocompatible properties, but often suffer from weak mechanical strength. Conventional strengthening strategies for protein-based hydrogels are to introduce nanoparticles or synthetic polymers for improving their mechanical strength, but often compromise their biocompatibility. Here, a new, general, protein unfolding-chemical coupling (PNC) strategy is developed to fabricate pure protein hydrogels without any additives to achieve both high mechanical strength and excellent cell biocompatibility. This PNC strategy combines thermal-induced protein unfolding/gelation to form a physically-crosslinked network and a -NH2/-COOH coupling reaction to generate a chemicallycrosslinked network. Using bovine serum albumin (BSA) as a globular protein, PNC-BSA hydrogels show macroscopic transparency, high stability, high mechanical properties (compressive/tensile strength of 115/0.43 MPa), fast stiffness/toughness recovery of 85%/91% at room temperature, good fatigue resistance, and low cell cytotoxicity and red blood cell hemolysis. More importantly, the PNC strategy can be not only generally applied to silk fibroin, ovalbumin, and milk albumin protein to form different, high strength protein hydrogels, but also modified with PEDOT/PSS nanoparticles as strain sensors and fluorescent fillers as color sensors. This work demonstrates a new, universal, PNC method to prepare high strength, multi-functional, pure protein hydrogels beyond a few available today.
Topics: Fibroins; Hydrogels; Polymers; Protein Unfolding; Serum Albumin, Bovine
PubMed: 34939355
DOI: 10.1002/advs.202102557 -
Small (Weinheim An Der Bergstrasse,... Sep 2017Spatiotemporal control of protein structure and activity in biological systems has important and broad implications in biomedical sciences as evidenced by recent...
Spatiotemporal control of protein structure and activity in biological systems has important and broad implications in biomedical sciences as evidenced by recent advances in optogenetic approaches. Here, this study demonstrates that nanosecond pulsed laser heating of gold nanoparticles (GNP) leads to an ultrahigh and ultrashort temperature increase, coined as "molecular hyperthermia", which causes selective unfolding and inactivation of proteins adjacent to the GNP. Protein inactivation is highly dependent on both laser pulse energy and GNP size, and has a well-defined impact zone in the nanometer scale. It is anticipated that the fine control over protein structure and function enabled by this discovery will be highly enabling within a number of arenas, from probing the biophysics of protein folding/unfolding to the nanoscopic manipulation of biological systems via an optical trigger, to developing novel therapeutics for disease treatment without genetic modification.
Topics: Gold; Hot Temperature; Metal Nanoparticles; Protein Unfolding; Proteins; Time Factors
PubMed: 28696524
DOI: 10.1002/smll.201700841 -
Biophysical Journal Jan 2016The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments...
The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale.
Topics: Bacterial Proteins; Biomechanical Phenomena; Elasticity; Kinetics; Mechanical Phenomena; Models, Molecular; Peptides; Protein Conformation; Protein Unfolding; Ribonucleases; Thermodynamics
PubMed: 26745410
DOI: 10.1016/j.bpj.2015.11.015 -
Analytical Chemistry May 2020Elucidating the structures and stabilities of proteins and their complexes is paramount to understanding their biological functions in cellular processes. Native mass...
Elucidating the structures and stabilities of proteins and their complexes is paramount to understanding their biological functions in cellular processes. Native mass spectrometry (MS) coupled with ion mobility spectrometry (IMS) is emerging as an important biophysical technique owing to its high sensitivity, rapid analysis time, and ability to interrogate sample complexity or heterogeneity and the ability to probe protein structure dynamics. Here, a commercial IMS-MS platform has been modified for static native ESI emitters and an extended mass-to-charge range (20 kDa /) and its performance capabilities and limits were explored for a range of protein and protein complexes. The results show new potential for this instrument platform for studies of large protein and protein complexes and provides a roadmap for extending the performance metrics for studies of even larger, more complex systems, namely, membrane protein complexes and their interactions with ligands.
Topics: Concanavalin A; Fructose-Bisphosphate Aldolase; Ion Mobility Spectrometry; Mass Spectrometry; Protein Conformation; Protein Unfolding; Streptavidin; Ubiquitin
PubMed: 32338885
DOI: 10.1021/acs.analchem.0c00772 -
Journal of Molecular Biology May 2016The AAA+ Cdc48 ATPase (alias p97 or VCP) is a key player in multiple ubiquitin-dependent cell signaling, degradation, and quality control pathways. Central to these... (Review)
Review
The AAA+ Cdc48 ATPase (alias p97 or VCP) is a key player in multiple ubiquitin-dependent cell signaling, degradation, and quality control pathways. Central to these broad biological functions is the ability of Cdc48 to interact with a large number of adaptor proteins and to remodel macromolecular proteins and their complexes. Different models have been proposed to explain how Cdc48 might couple ATP hydrolysis to forcible unfolding, dissociation, or remodeling of cellular clients. In this review, we provide an overview of possible mechanisms for substrate unfolding/remodeling by this conserved and essential AAA+ protein machine and their adaption and possible biological function throughout evolution.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Cell Cycle Proteins; Evolution, Molecular; Hydrolysis; Macromolecular Substances; Models, Biological; Models, Molecular; Protein Unfolding; Valosin Containing Protein
PubMed: 26608813
DOI: 10.1016/j.jmb.2015.11.015 -
ACS Nano Jul 2022Globular folded proteins are versatile nanoscale building blocks to create biomaterials with mechanical robustness and inherent biological functionality due to their...
Globular folded proteins are versatile nanoscale building blocks to create biomaterials with mechanical robustness and inherent biological functionality due to their specific and well-defined folded structures. Modulating the nanoscale unfolding of protein building blocks during network formation ( protein unfolding) provides potent opportunities to control the protein network structure and mechanics. Here, we control protein unfolding during the formation of hydrogels constructed from chemically cross-linked maltose binding protein using ligand binding and the addition of cosolutes to modulate protein kinetic and thermodynamic stability. Bulk shear rheology characterizes the storage moduli of the bound and unbound protein hydrogels and reveals a correlation between network rigidity, characterized as an increase in the storage modulus, and protein thermodynamic stability. Furthermore, analysis of the network relaxation behavior identifies a crossover from an unfolding dominated regime to an entanglement dominated regime. Control of protein unfolding and entanglement provides an important route to finely tune the architecture, mechanics, and dynamic relaxation of protein hydrogels. Such predictive control will be advantageous for future smart biomaterials for applications which require responsive and dynamic modulation of mechanical properties and biological function.
Topics: Hydrogels; Biocompatible Materials; Rheology; Proteins; Protein Unfolding
PubMed: 35731007
DOI: 10.1021/acsnano.2c02369