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Journal of Molecular Biology Jun 2023Single-molecule force spectroscopy is a unique method that can probe the structural changes of single proteins at a high spatiotemporal resolution while mechanically... (Review)
Review
Single-molecule force spectroscopy is a unique method that can probe the structural changes of single proteins at a high spatiotemporal resolution while mechanically manipulating them over a wide force range. Here, we review the current understanding of membrane protein folding learned by using the force spectroscopy approach. Membrane protein folding in lipid bilayers is one of the most complex biological processes in which diverse lipid molecules and chaperone proteins are intricately involved. The approach of single protein forced unfolding in lipid bilayers has produced important findings and insights into membrane protein folding. This review provides an overview of the forced unfolding approach, including recent achievements and technical advances. Progress in the methods can reveal more interesting cases of membrane protein folding and clarify general mechanisms and principles.
Topics: Lipid Bilayers; Membrane Proteins; Microscopy, Atomic Force; Protein Folding; Spectrum Analysis; Single Molecule Imaging
PubMed: 37330286
DOI: 10.1016/j.jmb.2023.167975 -
Methods in Molecular Biology (Clifton,... 2023Overexpression of heterologous protein in prokaryotic host cells, such as Escherichia coli, usually leads to formation of inactive and insoluble aggregates known as...
Overexpression of heterologous protein in prokaryotic host cells, such as Escherichia coli, usually leads to formation of inactive and insoluble aggregates known as inclusion bodies (IBs). Recovery of refolded and functionally bioactive proteins from IBs is a challenging task, and a unique condition (e.g., solubilizing and refolding buffers) for each individual protein should be experimentally obtained. Here, we present a simple protocol for development of solubilizing and refolding buffers for successful recovery of pure bioactive proteins from IBs.
Topics: Escherichia coli; Inclusion Bodies; Protein Refolding; Recombinant Proteins; Solubility
PubMed: 36656522
DOI: 10.1007/978-1-0716-2930-7_10 -
Science Advances Aug 2022SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S...
SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo-electron microscopy and cryo-electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo-electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein-directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.
Topics: Angiotensin-Converting Enzyme 2; COVID-19; Cryoelectron Microscopy; Humans; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Virus Internalization
PubMed: 35984891
DOI: 10.1126/sciadv.abo3153 -
Biomedicines Oct 2021While protein refolding has been studied for over 50 years since the pioneering work of Christian Anfinsen, there have been a limited number of studies correlating...
While protein refolding has been studied for over 50 years since the pioneering work of Christian Anfinsen, there have been a limited number of studies correlating results between chemical, thermal, and mechanical unfolding. The limited knowledge of the relationship between these processes makes it challenging to compare results between studies if different refolding methods were applied. Our current work compares the energetic barriers and folding rates derived from chemical, thermal, and mechanical experiments using an immunoglobulin-like domain from the muscle protein titin as a model system. This domain, I83, has high solubility and low stability relative to other Ig domains in titin, though its stability can be modulated by calcium. Our experiments demonstrated that the free energy of refolding was equivalent with all three techniques, but the refolding rates exhibited differences, with mechanical refolding having slightly faster rates. This suggests that results from equilibrium-based measurements can be compared directly but care should be given comparing refolding kinetics derived from refolding experiments that used different unfolding methods.
PubMed: 34680512
DOI: 10.3390/biomedicines9101395 -
Zhejiang Da Xue Xue Bao. Yi Xue Ban =... Nov 2022Molecular chaperones and co-chaperones facilitate the assembly of newly synthesized polypeptides and refolding of unfolded or misfolded proteins, thereby maintaining... (Review)
Review
Molecular chaperones and co-chaperones facilitate the assembly of newly synthesized polypeptides and refolding of unfolded or misfolded proteins, thereby maintaining protein homeostasis in cells. As co-chaperones of the master chaperone heat shock protein (HSP) 70, the HSP40 (DNAJ) proteins are largest chaperone family in eukaryotic cells. They contain a characteristic J-domain which mediates interaction with HSP70, thereby helping protein folding. It is well perceived that protein homeostasis is vital for neuronal health. DNAJ family proteins have been linked to the occurrence and progression of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, Charcot-Marie-Tooth disease, spinal muscular atrophy, distal hereditary motor neuropathy, limb-girdle type muscular dystrophy, neuronal ceroid lipofuscinosis and essential tremor in recent studies. DNAJA1 effectively degrades huntington aggregates; DNAJB1 can degrade protein aggregates ataxin-3; DNAJB2 can inhibit the formation of huntington aggregates; DNAJB6 can inhibit the aggregation of Aβ and α-synuclein; DNAJC5 can promote the release of TDP-43, τ protein, and α-synuclein into the extracellular space. Mutations in the essential tremor-associated DNAJC13 gene can prevent endosome protein trafficking. This article reviews the mechanism of DNAJ protein family in neurodegenerative diseases.
Topics: Humans; HSP40 Heat-Shock Proteins; alpha-Synuclein; Neurodegenerative Diseases; Essential Tremor; Protein Folding; Nerve Tissue Proteins; Molecular Chaperones
PubMed: 36581576
DOI: 10.3724/zdxbyxb-2021-0406 -
Experimental Cell Research Feb 2021HSP70 chaperones, J-domain proteins (JDPs) and nucleotide exchange factors (NEF) form functional networks that have the ability to prevent and reverse the aggregation of... (Review)
Review
HSP70 chaperones, J-domain proteins (JDPs) and nucleotide exchange factors (NEF) form functional networks that have the ability to prevent and reverse the aggregation of proteins associated with neurodegenerative diseases. JDPs can interact with specific substrate proteins, hold them in a refolding-competent conformation and target them to specific HSP70 chaperones for remodeling. Thereby, JDPs select specific substrates and constitute an attractive target for pharmacological intervention of neurodegenerative diseases. This, under the condition that the exact mechanism of JDPs interaction with specific substrates is unveiled. In this review, we provide an overview of the structural and functional variety of JDPs that interact with neurodegenerative disease-associated proteins and we highlight those studies that identified specific residues, domains or regions of JDPs that are crucial for substrate binding.
Topics: Animals; Carrier Proteins; HSP70 Heat-Shock Proteins; Humans; Molecular Chaperones; Neurodegenerative Diseases; Protein Binding; Protein Folding; Protein Interaction Domains and Motifs; Protein Interaction Maps
PubMed: 33460589
DOI: 10.1016/j.yexcr.2021.112491 -
Biochimica Et Biophysica Acta.... Mar 2022Heat Shock Proteins (HSPs) and their co-chaperones have well-established roles in regulating proteostasis within the cell, the nature of which continues to emerge with... (Review)
Review
Heat Shock Proteins (HSPs) and their co-chaperones have well-established roles in regulating proteostasis within the cell, the nature of which continues to emerge with further study. To date, HSPs have been shown to be integral to protein folding and re-folding, protein transport, avoidance of protein aggregation, and modulation of protein degradation. Many cell signaling events are mediated by the chemical modification of proteins post-translationally that can alter protein conformation and activity, although it is not yet known whether the changes in protein conformation induced by post-translational modifications (PTMs) are also dependent upon HSPs and their co-chaperones for subsequent protein re-folding. We discuss what is known regarding roles for HSPs and other molecular chaperones in cell signaling events with a focus on oncogenic signaling. We also propose a hypothesis by which Hsp70 and Hsp90 may co-operate to facilitate cell signaling events that may link PTMs with the cellular protein folding machinery.
Topics: Animals; Heat-Shock Proteins; Humans; Molecular Chaperones; Neoplasms; Proteostasis; Signal Transduction
PubMed: 34906617
DOI: 10.1016/j.bbamcr.2021.119187 -
Applied Microbiology and Biotechnology Mar 2021Overexpression of recombinant proteins in Escherichia coli results in misfolded and non-active protein aggregates in the cytoplasm, so-called inclusion bodies (IB). In... (Review)
Review
Overexpression of recombinant proteins in Escherichia coli results in misfolded and non-active protein aggregates in the cytoplasm, so-called inclusion bodies (IB). In recent years, a change in the mindset regarding IBs could be observed: IBs are no longer considered an unwanted waste product, but a valid alternative to produce a product with high yield, purity, and stability in short process times. However, solubilization of IBs and subsequent refolding is necessary to obtain a correctly folded and active product. This protein refolding process is a crucial downstream unit operation-commonly done as a dilution in batch or fed-batch mode. Drawbacks of the state-of-the-art include the following: the large volume of buffers and capacities of refolding tanks, issues with uniform mixing, challenging analytics at low protein concentrations, reaction kinetics in non-usable aggregates, and generally low re-folding yields. There is no generic platform procedure available and a lack of robust control strategies. The introduction of Quality by Design (QbD) is the method-of-choice to provide a controlled and reproducible refolding environment. However, reliable online monitoring techniques to describe the refolding kinetics in real-time are scarce. In our view, only monitoring and control of re-folding kinetics can ensure a productive, scalable, and versatile platform technology for re-folding processes. For this review, we screened the current literature for a combination of online process analytical technology (PAT) and modeling techniques to ensure a controlled refolding process. Based on our research, we propose an integrated approach based on the idea that all aspects that cannot be monitored directly are estimated via digital twins and used in real-time for process control. KEY POINTS: • Monitoring and a thorough understanding of refolding kinetics are essential for model-based control of refolding processes. • The introduction of Quality by Design combining Process Analytical Technology and modeling ensures a robust platform for inclusion body refolding.
Topics: Inclusion Bodies; Kinetics; Protein Folding; Protein Refolding; Recombinant Proteins; Technology
PubMed: 33598720
DOI: 10.1007/s00253-021-11151-y -
Methods in Molecular Biology (Clifton,... 2022Expression of heterologous proteins in E. coli often leads to the formation of protein aggregates known as inclusion bodies (IBs). Inclusion body aggregates pose a major...
Expression of heterologous proteins in E. coli often leads to the formation of protein aggregates known as inclusion bodies (IBs). Inclusion body aggregates pose a major hurdle in the recovery of bioactive proteins from E. coli. Usage of strong denaturing buffers for solubilization of bacterial IBs results in poor recovery of bioactive protein. Structure-function understanding of IBs in the last two decades have led to the development of several mild solubilization buffers, which improve the recovery of bioactive from IBs. Recently, combinatorial mild solubilization methods have paved the way for solubilization of wide range of inclusion bodies with appreciable refolding yield. Here, we describe a simple protocol for solubilization and refolding of an inclusion body protein with appreciable recovery.
Topics: Escherichia coli; Inclusion Bodies; Protein Refolding; Proteins; Recombinant Proteins; Solubility
PubMed: 35089569
DOI: 10.1007/978-1-0716-1859-2_22 -
Journal of Alzheimer's Disease : JAD 2022Specific protein misfolding and aggregation are mechanisms underlying various neurodegenerative diseases such as prion disease and Alzheimer's disease (AD). The... (Review)
Review
Specific protein misfolding and aggregation are mechanisms underlying various neurodegenerative diseases such as prion disease and Alzheimer's disease (AD). The misfolded proteins are involved in prions, amyloid-β (Aβ), tau, and α-synuclein disorders; they share common structural, biological, and biochemical characteristics, as well as similar mechanisms of aggregation and self-propagation. Pathological features of AD include the appearance of plaques consisting of deposition of protein Aβ and neurofibrillary tangles formed by the hyperphosphorylated tau protein. Although it is not clear how protein aggregation leads to AD, we are learning that the cellular prion protein (PrPC) plays an important role in the pathogenesis of AD. Herein, we first examined the pathogenesis of prion and AD with a focus on the contribution of PrPC to the development of AD. We analyzed the mechanisms that lead to the formation of a high affinity bond between Aβ oligomers (AβOs) and PrPC. Also, we studied the role of PrPC as an AβO receptor that initiates an AβO-induced signal cascade involving mGluR5, Fyn, Pyk2, and eEF2K linking Aβ and tau pathologies, resulting in the death of neurons in the central nervous system. Finally, we have described how the PrPC-AβOs interaction can be used as a new potential therapeutic target for the treatment of PrPC-dependent AD.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Humans; Neurofibrillary Tangles; Neurons; Prion Proteins; Protein Aggregation, Pathological; Randomized Controlled Trials as Topic; Receptor, Metabotropic Glutamate 5; alpha-Synuclein; tau Proteins
PubMed: 34864675
DOI: 10.3233/JAD-215171