-
Frontiers in Molecular Biosciences 2022Human health depends on the correct folding of proteins, for misfolding and aggregation lead to diseases. An unfolded (denatured) protein can refold to its original...
Human health depends on the correct folding of proteins, for misfolding and aggregation lead to diseases. An unfolded (denatured) protein can refold to its original folded state. How does this occur is known as the protein folding problem. One of several related questions to this problem is that how much more stable is the folded state than the unfolded state. There are several measures of protein stability. In this article, protein stability is given a thermodynamic definition and is measured by Gibbs free energy change ( ) associated with the equilibrium, native (N) conformation ↔ denatured (D) conformation under the physiological condition usually taken as dilute buffer (or water) at 25 °C. We show that this thermodynamic quantity ( ), where subscript D represents transition between N and D states, and superscript 0 (zero) represents the fact that the transition occurs in the absence of denaturant, can be neither measured nor predicted under physiological conditions. However, can be measured in the presence of strong chemical denaturants such as guanidinium chloride and urea which are shown to destroy all noncovalent interactions responsible for maintaining the folded structure. A problem with this measurement is that the estimate of comes from the analysis of the plot of denaturant concentration, which requires a long extrapolation of values of , and all the three methods of extrapolation give three different values of for a protein. Thus, our confidence in the authentic value of is eroded. Another problem with this measurement of is that it is done on the pure protein sample in dilute buffer which is a very large extrapolation of the conditions, for the crowding effect on protein stability is ignored.
PubMed: 35992266
DOI: 10.3389/fmolb.2022.880358 -
Pharmaceutics Nov 2022Maintaining the structure of protein and peptide drugs has become one of the most important goals of scientists in recent decades. Cold and thermal denaturation... (Review)
Review
Maintaining the structure of protein and peptide drugs has become one of the most important goals of scientists in recent decades. Cold and thermal denaturation conditions, lyophilization and freeze drying, different pH conditions, concentrations, ionic strength, environmental agitation, the interaction between the surface of liquid and air as well as liquid and solid, and even the architectural structure of storage containers are among the factors that affect the stability of these therapeutic biomacromolecules. The use of genetic engineering, side-directed mutagenesis, fusion strategies, solvent engineering, the addition of various preservatives, surfactants, and additives are some of the solutions to overcome these problems. This article will discuss the types of stress that lead to instabilities of different proteins used in pharmaceutics including regulatory proteins, antibodies, and antibody-drug conjugates, and then all the methods for fighting these stresses will be reviewed. New and existing analytical methods that are used to detect the instabilities, mainly changes in their primary and higher order structures, are briefly summarized.
PubMed: 36432723
DOI: 10.3390/pharmaceutics14112533 -
Angewandte Chemie (International Ed. in... Sep 2021Cells transmit piconewton forces to receptors to mediate processes such as migration and immune recognition. A major challenge in quantifying such forces is the sparsity...
Cells transmit piconewton forces to receptors to mediate processes such as migration and immune recognition. A major challenge in quantifying such forces is the sparsity of cell mechanical events. Accordingly, molecular tension is typically quantified with high resolution fluorescence microscopy, which hinders widespread adoption and application. Here, we report a mechanically triggered hybridization chain reaction (mechano-HCR) that allows chemical amplification of mechanical events. The amplification is triggered when a cell receptor mechanically denatures a duplex revealing a cryptic initiator to activate the HCR reaction in situ. Importantly, mechano-HCR enables direct readout of pN forces using a plate reader. We leverage this capability and measured mechano-IC for aspirin, Y-27632, and eptifibatide. Given that cell mechanical phenotypes are of clinical importance, mechano-HCR may offer a convenient route for drug discovery, personalized medicine, and disease diagnosis.
Topics: Aspirin; Eptifibatide; Humans; Nucleic Acid Hybridization
PubMed: 34242462
DOI: 10.1002/anie.202107660 -
Protein Science : a Publication of the... Sep 2021Seventy years ago, we learned from Chris Anfinsen that the stereochemical code necessary to fold a protein is embedded into its amino acid sequence. In water, protein... (Review)
Review
Seventy years ago, we learned from Chris Anfinsen that the stereochemical code necessary to fold a protein is embedded into its amino acid sequence. In water, protein morphogenesis is a spontaneous reversible process leading from an ensemble of disordered structures to the ordered functionally competent protein; conforming to Aristotle's definition of substance, the synolon of matter and form. The overall process of folding is generally consistent with a two state transition between the native and the denatured protein: not only the denatured state is an ensemble of several structures, but also the native protein populates distinct functionally relevant conformational (sub)states. This two-state view should be revised, given that any globular protein can populate a peculiar third state called amyloid, characterized by an overall architecture that at variance with the native state, is by-and-large independent of the primary structure. In a nut shell, we should accept that beside the folded and unfolded states, any protein can populate a third state called amyloid which gained center stage being the hallmark of incurable neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases as well as others. These fatal diseases are characterized by clear-cut clinical differences, yet display some commonalities such as the presence in the brain of amyloid deposits constituted by one misfolded protein specific for each disease. Some aspects of this complex problem are summarized here as an excursus from the prion's fibrils observed in the brain of aborigines who died of Kuru to the amyloid detectable in the cortex of Alzheimer's patients.
Topics: Alzheimer Disease; Amyloid; Amyloid beta-Protein Precursor; Brain; Gene Expression; Humans; Kuru; Models, Molecular; Parkinson Disease; PrPC Proteins; PrPSc Proteins; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Denaturation; Protein Folding; Thermodynamics; alpha-Synuclein; tau Proteins
PubMed: 34118168
DOI: 10.1002/pro.4145 -
Journal of Proteome Research Feb 2023The structure of a protein defines its function and integrity and correlates with the protein folding stability (PFS). Quantifying PFS allows researchers to assess...
The structure of a protein defines its function and integrity and correlates with the protein folding stability (PFS). Quantifying PFS allows researchers to assess differential stability of proteins in different disease or ligand binding states, providing insight into protein efficacy and potentially serving as a metric of protein quality. There are a number of mass spectrometry (MS)-based methods to assess PFS, such as hermal rotein rofiling (TPP), tability of roteins from ates of idation (SPROX), and odination rotein tability ssay (IPSA). Despite the critical value that PFS studies add to the understanding of mechanisms of disease and treatment development, proteomics research is still primarily dominated by concentration-based studies. We found that a major reason for the lack of PFS studies is the lack of a user-friendly data processing tool. Here we present the first user-friendly software, C with a graphical user interface for calculating PFS. Besides calculating site-specific PFS of a given protein from chemical denature folding stability assays, C is also compatible with thermal denature folding stability assays. C also includes a set of data visualization tools to help identify changes in PFS across protein sequences and in between different treatment conditions. We expect the introduction of C to lower the barrier of entry for researchers to investigate PFS, promoting the usage of PFS in studies. In the long run, we expect this increase in PFS research to accelerate our understanding of the pathogenesis and pathophysiology of disease.
Topics: Proteins; Mass Spectrometry; Protein Stability; Amino Acid Sequence; Software; Protein Folding
PubMed: 36707058
DOI: 10.1021/acs.jproteome.2c00619 -
Open Biology Jul 2022How alpha-helical membrane proteins fold correctly in the highly hydrophobic membrane interior is not well understood. Their folding is known to be highly influenced by... (Review)
Review
How alpha-helical membrane proteins fold correctly in the highly hydrophobic membrane interior is not well understood. Their folding is known to be highly influenced by the lipids within the surrounding bilayer, but the majority of folding studies have focused on detergent-solubilized protein rather than protein in a lipid environment. There are different ways to study folding in lipid bilayers, and each method has its own advantages and disadvantages. This review will discuss folding methods which can be used to study alpha-helical membrane proteins in bicelles, liposomes, nanodiscs or native membranes. These folding methods include folding methods in liposomes such as denaturant unfolding studies, and single-molecule force spectroscopy studies in bicelles, liposomes and native membranes. This review will also discuss recent advances in co-translational folding studies, which use cell-free expression with liposomes or nanodiscs or are performed with native membranes.
Topics: Lipid Bilayers; Liposomes; Membrane Proteins; Protein Conformation, alpha-Helical; Protein Folding
PubMed: 35855589
DOI: 10.1098/rsob.220054 -
Nature Methods Sep 2022Antibodies have diverse applications due to their high reaction specificities but are sensitive to denaturation when a higher working temperature is required. We have...
Antibodies have diverse applications due to their high reaction specificities but are sensitive to denaturation when a higher working temperature is required. We have developed a simple, highly scalable and generalizable chemical approach for stabilizing off-the-shelf antibodies against thermal and chemical denaturation. We demonstrate that the stabilized antibodies (termed SPEARs) can withstand up to 4 weeks of continuous heating at 55 °C and harsh denaturants, and apply our method to 33 tested antibodies. SPEARs enable flexible applications of thermocycling and denaturants to dynamically modulate their binding kinetics, reaction equilibrium, macromolecular diffusivity and aggregation propensity. In particular, we show that SPEARs permit the use of a thermally facilitated three-dimensional immunolabeling strategy (termed ThICK staining), achieving whole mouse brain immunolabeling within 72 h, as well as nearly fourfold deeper penetration with threefold less antibodies in human brain tissue. With faster deep-tissue immunolabeling and broad compatibility with tissue processing and clearing methods without the need for any specialized equipment, we anticipate the wide applicability of ThICK staining with SPEARs for deep immunostaining.
Topics: Animals; Antibodies; Brain; Humans; Mice
PubMed: 36050489
DOI: 10.1038/s41592-022-01569-1 -
Nature Methods Dec 2022We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that...
We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-Å resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 Å) and an mGreenLantern mutant (1.2 Å), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.
Topics: Luminescent Proteins; Microscopy; Green Fluorescent Proteins; Fluorescence Resonance Energy Transfer; Light
PubMed: 36344833
DOI: 10.1038/s41592-022-01660-7 -
Molecules (Basel, Switzerland) Oct 2022The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be... (Review)
Review
The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be caused by chemical and physical agents due to changes in the physicochemical conditions of pH, ionic strength, temperature, pressure, and electric field or due to the presence of a cosolvent that perturbs the delicate balance between stabilizing and destabilizing interactions and eventually induces chemical modifications. For most proteins, denaturation is a complex process involving transient intermediates in several reversible and eventually irreversible steps. Knowledge of protein stability and denaturation processes is mandatory for the development of enzymes as industrial catalysts, biopharmaceuticals, analytical and medical bioreagents, and safe industrial food. Electrophoresis techniques operating under extreme conditions are convenient tools for analyzing unfolding transitions, trapping transient intermediates, and gaining insight into the mechanisms of denaturation processes. Moreover, quantitative analysis of electrophoretic mobility transition curves allows the estimation of the conformational stability of proteins. These approaches include polyacrylamide gel electrophoresis and capillary zone electrophoresis under cold, heat, and hydrostatic pressure and in the presence of non-ionic denaturing agents or stabilizers such as polyols and heavy water. Lastly, after exposure to extremes of physical conditions, electrophoresis under standard conditions provides information on irreversible processes, slow conformational drifts, and slow renaturation processes. The impressive developments of enzyme technology with multiple applications in fine chemistry, biopharmaceutics, and nanomedicine prompted us to revisit the potentialities of these electrophoretic approaches. This feature review is illustrated with published and unpublished results obtained by the authors on cholinesterases and paraoxonase, two physiologically and toxicologically important enzymes.
Topics: Protein Denaturation; Protein Conformation; Deuterium Oxide; Aryldialkylphosphatase; Electrophoresis, Polyacrylamide Gel; Cholinesterases; Biological Products; Thermodynamics; Protein Folding
PubMed: 36296453
DOI: 10.3390/molecules27206861 -
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