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Microbial Cell Factories Mar 2015Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from... (Review)
Review
Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by optimizing the individual steps of the process, especially solubilization of the inclusion bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in inclusion body aggregates have native-like structure. These methods solubilize the inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from inclusion bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.
Topics: Escherichia coli; Inclusion Bodies; Models, Molecular; Protein Denaturation; Protein Folding; Protein Refolding; Protein Unfolding; Recombinant Proteins; Solubility
PubMed: 25889252
DOI: 10.1186/s12934-015-0222-8 -
Annual Review of Biophysics May 2023My accidental encounter with protein hydrogen exchange (HX) at its very beginning and its continued development through my scientific career have led us to a series of... (Review)
Review
My accidental encounter with protein hydrogen exchange (HX) at its very beginning and its continued development through my scientific career have led us to a series of advances in HX measurement, interpretation, and cutting edge biophysical applications. After some thoughts about how life brought me there, I take the opportunity to reflect on our early studies of allosteric structure and energy change in hemoglobin, the still-current protein folding problem, and our most recent forward-looking studies on protein machines.
Topics: Biophysics; Protein Folding
PubMed: 36630583
DOI: 10.1146/annurev-biophys-062122-093517 -
Protein Science : a Publication of the... Jul 2020The intriguing process of protein folding comprises discrete steps that stabilize the protein molecules in different conformations. The metastable state of protein is... (Review)
Review
The intriguing process of protein folding comprises discrete steps that stabilize the protein molecules in different conformations. The metastable state of protein is represented by specific conformational characteristics, which place the protein in a local free energy minimum state of the energy landscape. The native-to-metastable structural transitions are governed by transient or long-lived thermodynamic and kinetic fluctuations of the intrinsic interactions of the protein molecules. Depiction of the structural and functional properties of metastable proteins is not only required to understand the complexity of folding patterns but also to comprehend the mechanisms of anomalous aggregation of different proteins. In this article, we review the properties of metastable proteins in context of their stability and capability of undergoing atypical aggregation in physiological conditions.
Topics: Kinetics; Models, Molecular; Protein Conformation; Protein Folding; Proteins; Thermodynamics
PubMed: 32223005
DOI: 10.1002/pro.3859 -
Disease Models & Mechanisms Jan 2014From unicellular organisms to humans, cells have evolved elegant systems to facilitate careful folding of proteins and the maintenance of protein homeostasis. Key...
From unicellular organisms to humans, cells have evolved elegant systems to facilitate careful folding of proteins and the maintenance of protein homeostasis. Key modulators of protein homeostasis include a large, conserved family of proteins known as molecular chaperones, which augment the folding of nascent polypeptides and temper adverse consequences of cellular stress. However, errors in protein folding can still occur, resulting in the accumulation of misfolded proteins that strain cellular quality-control systems. In some cases, misfolded proteins can be targeted for degradation by the proteasome or via autophagy. Nevertheless, protein misfolding is a feature of many complex, genetically and clinically pleiotropic diseases, including neurodegenerative disorders and cancer. In recent years, substantial progress has been made in unraveling the complexity of protein folding using model systems, and we are now closer to being able to diagnose and treat the growing number of protein-folding diseases. To showcase some of these important recent advances, and also to inspire discussion on approaches to tackle unanswered questions, Disease Models & Mechanisms (DMM) presents a special collection of reviews from researchers at the cutting-edge of the field.
Topics: Animals; Autophagy; Genetic Diseases, Inborn; Humans; Neoplasms; Neurodegenerative Diseases; Proteasome Endopeptidase Complex; Protein Conformation; Protein Denaturation; Protein Folding; Proteostasis Deficiencies
PubMed: 24396147
DOI: 10.1242/dmm.014985 -
ACS Chemical Biology Aug 2019A complete inventory of the forces governing protein folding is critical for productive protein modeling, including structure prediction and design, as well as... (Review)
Review
A complete inventory of the forces governing protein folding is critical for productive protein modeling, including structure prediction and design, as well as understanding protein misfolding diseases of clinical significance. The dominant contributors to protein folding include the hydrophobic effect and conventional hydrogen bonding, along with Coulombic and van der Waals interactions. Over the past few decades, important additional contributors have been identified, including C-H···O hydrogen bonding, →π* interactions, C5 hydrogen bonding, chalcogen bonding, and interactions involving aromatic rings (cation-π, X-H···π, π-π, anion-π, and sulfur-arene). These secondary contributions fall into two general classes: (1) weak but abundant interactions of the protein main chain and (2) strong but less frequent interactions involving protein side chains. Though interactions with high individual energies play important roles in specifying nonlocal molecular contacts and ligand binding, we estimate that weak but abundant interactions are likely to make greater overall contributions to protein folding, particularly at the level of secondary structure. Further research is likely to illuminate additional roles of these noncanonical interactions and could also reveal contributions yet unknown.
Topics: Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Protein Folding; Proteins; Static Electricity
PubMed: 31243961
DOI: 10.1021/acschembio.9b00339 -
Current Opinion in Structural Biology Aug 2021Membrane proteins have historically been recalcitrant to biophysical folding studies. However, recent adaptations of methods from the soluble protein folding field have... (Review)
Review
Membrane proteins have historically been recalcitrant to biophysical folding studies. However, recent adaptations of methods from the soluble protein folding field have found success in their applications to transmembrane proteins composed of both α-helical and β-barrel conformations. Avoiding aggregation is critical for the success of these experiments. Altogether these studies are leading to discoveries of folding trajectories, foundational stabilizing forces and better-defined endpoints that enable more accurate interpretation of thermodynamic data. Increased information on membrane protein folding in the cell shows that the emerging biophysical principles are largely recapitulated even in the complex biological environment.
Topics: Membrane Proteins; Protein Folding; Thermodynamics
PubMed: 33975156
DOI: 10.1016/j.sbi.2021.03.006 -
Proceedings of the National Academy of... Aug 2022Repeat proteins are made with tandem copies of similar amino acid stretches that fold into elongated architectures. These proteins constitute excellent model systems to...
Repeat proteins are made with tandem copies of similar amino acid stretches that fold into elongated architectures. These proteins constitute excellent model systems to investigate how evolution relates to structure, folding, and function. Here, we propose a scheme to map evolutionary information at the sequence level to a coarse-grained model for repeat-protein folding and use it to investigate the folding of thousands of repeat proteins. We model the energetics by a combination of an inverse Potts-model scheme with an explicit mechanistic model of duplications and deletions of repeats to calculate the evolutionary parameters of the system at the single-residue level. These parameters are used to inform an Ising-like model that allows for the generation of folding curves, apparent domain emergence, and occupation of intermediate states that are highly compatible with experimental data in specific case studies. We analyzed the folding of thousands of natural Ankyrin repeat proteins and found that a multiplicity of folding mechanisms are possible. Fully cooperative all-or-none transitions are obtained for arrays with enough sequence-similar elements and strong interactions between them, while noncooperative element-by-element intermittent folding arose if the elements are dissimilar and the interactions between them are energetically weak. Additionally, we characterized nucleation-propagation and multidomain folding mechanisms. We show that the global stability and cooperativity of the repeating arrays can be predicted from simple sequence scores.
Topics: Ankyrin Repeat; Models, Chemical; Protein Folding
PubMed: 35905321
DOI: 10.1073/pnas.2204131119 -
Biomolecules Apr 2022Protein folding and structural biology are highly active disciplines that combine basic research in various fields, including biology, chemistry, physics, and computer...
Protein folding and structural biology are highly active disciplines that combine basic research in various fields, including biology, chemistry, physics, and computer science, with practical applications in biomedicine and nanotechnology. However, there are still gaps in the understanding of the detailed mechanisms of protein folding, and protein structure-function relations. In an effort to bridge these gaps, this paper studies the equivalence of proteins and origami. Research on proteins and origami provides strong evidence to support the use of origami folding principles and mechanical models to explain aspects of proteins formation and function. Although not identical, the equivalence of origami and proteins emerges in: (i) the folding processes, (ii) the shape and structure of proteins and origami models, and (iii) the intrinsic mechanical properties of the folded structures/models, which allows them to synchronically fold/unfold and effectively distribute forces to the whole structure. As a result, origami can contribute to the understanding of various key protein-related mechanisms and support the design of de novo proteins and nanomaterials.
Topics: Nanostructures; Nanotechnology; Protein Folding; Proteins
PubMed: 35625549
DOI: 10.3390/biom12050622 -
Protein Science : a Publication of the... Nov 2016A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free... (Review)
Review
A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two-state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non-cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier-less "downhill" folding, as well as for continuous "uphill" unfolding transitions, indicate that gradual non-cooperative processes may be ubiquitous features on the free energy landscape of protein folding.
Topics: Models, Chemical; Models, Molecular; Protein Folding; Thermodynamics
PubMed: 27522064
DOI: 10.1002/pro.3015 -
Current Opinion in Structural Biology Feb 2022Delineating the folding steps of helical-bundle membrane proteins has been a challenging task. Many questions remain unanswered, including the conformation and stability... (Review)
Review
Delineating the folding steps of helical-bundle membrane proteins has been a challenging task. Many questions remain unanswered, including the conformation and stability of the states populated during folding, the shape of the energy barriers between the states, and the role of lipids as a solvent in mediating the folding. Recently, theoretical frames have matured to a point that permits detailed dissection of the folding steps, and advances in experimental techniques at both single-molecule and ensemble levels enable selective modulation of specific steps for quantitative determination of the folding energy landscapes. We also discuss how lipid molecules would play an active role in shaping the folding energy landscape of membrane proteins, and how folding of multi-domain membrane proteins can be understood based on our current knowledge. We conclude this review by offering an outlook for emerging questions in the study of membrane protein folding.
Topics: Membrane Proteins; Protein Folding; Thermodynamics
PubMed: 34995926
DOI: 10.1016/j.sbi.2021.11.013