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Advances in Colloid and Interface... Oct 2022Although the anionic surfactant sodium dodecyl sulfate, SDS, has been used for more than half a century as a versatile and efficient protein denaturant for protein... (Review)
Review
Although the anionic surfactant sodium dodecyl sulfate, SDS, has been used for more than half a century as a versatile and efficient protein denaturant for protein separation and size estimation, there is still controversy about its mode of interaction with proteins. The term "rod-like" structures for the complexes that form between SDS and protein, originally introduced by Tanford, is not sufficiently descriptive and does not distinguish between the two current vying models, namely protein-decorated micelles a.k.a. the core-shell model (in which denatured protein covers the surface of micelles) versus beads-on-a-string model (where unfolded proteins are surrounded by surfactant micelles). Thanks to a combination of structural, kinetic and computational work particularly within the last 5-10 years, it is now possible to rule decisively in favor of the core-shell model. This is supported unambiguously by a combination of calorimetric and small-angle X-ray scattering (SAXS) techniques and confirmed by increasingly sophisticated molecular dynamics simulations. Depending on the SDS:protein ratio and the protein molecular mass, the formed structures can range from multiple partly unfolded protein molecules surrounding a single shared micelle to a single polypeptide chain decorating multiple micelles. We also have much new insight into how this species forms. It is preceded by the binding of small numbers of SDS molecules which subsequently grow by accretion. Time-resolved SAXS analysis reveals an asymmetric attack by SDS micelles followed by distribution of the increasingly unfolded protein around the micelle. The compactness of the protein chain continues to evolve at higher SDS concentrations according to single-molecule studies, though the protein remains completely denatured on the tertiary structural level. SDS denaturation can be reversed by addition of nonionic surfactants that absorb SDS forming mixed micelles, leaving the protein free to refold. Refolding can occur in parallel tracks if only a fraction of the protein is initially stripped of SDS. SDS unfolding is nearly always reversible unless carried out at low pH, where charge neutralization can lead to superclusters of protein-surfactant complexes. With the general mechanism of SDS denaturation now firmly established, it largely remains to explore how other ionic surfactants (including biosurfactants) may diverge from this path.
Topics: Micelles; Proteins; Scattering, Small Angle; Sodium Dodecyl Sulfate; Surface-Active Agents; X-Ray Diffraction
PubMed: 36027673
DOI: 10.1016/j.cis.2022.102754 -
International Journal of Molecular... Feb 2024Proteins are large biomolecules with a specific structure that is composed of one or more long amino acid chains. Correct protein structures are directly linked to their... (Review)
Review
Proteins are large biomolecules with a specific structure that is composed of one or more long amino acid chains. Correct protein structures are directly linked to their correct function, and many environmental factors can have either positive or negative effects on this structure. Thus, there is a clear need for methods enabling the study of proteins, their correct folding, and components affecting protein stability. There is a significant number of label-free methods to study protein stability. In this review, we provide a general overview of these methods, but the main focus is on fluorescence-based low-instrument and -expertise-demand techniques. Different aspects related to thermal shift assays (TSAs), also called differential scanning fluorimetry (DSF) or ThermoFluor, are introduced and compared to isothermal chemical denaturation (ICD). Finally, we discuss the challenges and comparative aspects related to these methods, as well as future opportunities and assay development directions.
Topics: Protein Stability; Proteins; Amino Acids; Fluorometry; Biological Assay; Protein Denaturation
PubMed: 38339045
DOI: 10.3390/ijms25031764 -
Biological Chemistry Dec 2019Proteasomes are the principal molecular machines for the regulated degradation of intracellular proteins. These self-compartmentalized macromolecular assemblies... (Review)
Review
Proteasomes are the principal molecular machines for the regulated degradation of intracellular proteins. These self-compartmentalized macromolecular assemblies selectively degrade misfolded, mistranslated, damaged or otherwise unwanted proteins, and play a pivotal role in the maintenance of cellular proteostasis, in stress response, and numerous other processes of vital importance. Whereas the molecular architecture of the proteasome core particle (CP) is universally conserved, the unfoldase modules vary in overall structure, subunit complexity, and regulatory principles. Proteasomal unfoldases are AAA+ ATPases (ATPases associated with a variety of cellular activities) that unfold protein substrates, and translocate them into the CP for degradation. In this review, we summarize the current state of knowledge about proteasome - unfoldase systems in bacteria, archaea, and eukaryotes, the three domains of life.
Topics: ATPases Associated with Diverse Cellular Activities; Archaea; Bacteria; Molecular Chaperones; Proteasome Endopeptidase Complex; Protein Unfolding; Proteolysis; Proteostasis; Stress, Physiological
PubMed: 31665105
DOI: 10.1515/hsz-2019-0344 -
Nature Sep 2021Protein quality control systems are crucial for cellular function and organismal health. At present, most known protein quality control systems are multicomponent...
Protein quality control systems are crucial for cellular function and organismal health. At present, most known protein quality control systems are multicomponent machineries that operate via ATP-regulated interactions with non-native proteins to prevent aggregation and promote folding, and few systems that can broadly enable protein folding by a different mechanism have been identified. Moreover, proteins that contain the extensively charged poly-Asp/Glu (polyD/E) region are common in eukaryotic proteomes, but their biochemical activities remain undefined. Here we show that DAXX, a polyD/E protein that has been implicated in diverse cellular processes, possesses several protein-folding activities. DAXX prevents aggregation, solubilizes pre-existing aggregates and unfolds misfolded species of model substrates and neurodegeneration-associated proteins. Notably, DAXX effectively prevents and reverses aggregation of its in vivo-validated client proteins, the tumour suppressor p53 and its principal antagonist MDM2. DAXX can also restore native conformation and function to tumour-associated, aggregation-prone p53 mutants, reducing their oncogenic properties. These DAXX activities are ATP-independent and instead rely on the polyD/E region. Other polyD/E proteins, including ANP32A and SET, can also function as stand-alone, ATP-independent molecular chaperones, disaggregases and unfoldases. Thus, polyD/E proteins probably constitute a multifunctional protein quality control system that operates via a distinctive mechanism.
Topics: Animals; Cell Line; Cells; Co-Repressor Proteins; Evolution, Molecular; Humans; Models, Molecular; Molecular Chaperones; Mutation; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation; Protein Domains; Protein Folding; Protein Unfolding; Proteostasis Deficiencies; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53
PubMed: 34408321
DOI: 10.1038/s41586-021-03824-5 -
Cold Spring Harbor Perspectives in... Jan 2020Cells invest in an extensive network of factors to maintain protein homeostasis (proteostasis) and prevent the accumulation of potentially toxic protein aggregates. This... (Review)
Review
Cells invest in an extensive network of factors to maintain protein homeostasis (proteostasis) and prevent the accumulation of potentially toxic protein aggregates. This proteostasis network (PN) comprises the machineries for the biogenesis, folding, conformational maintenance, and degradation of proteins with molecular chaperones as central coordinators. Here, we review recent progress in understanding the modular architecture of the PN in mammalian cells and how it is modified during cell differentiation. We discuss the capacity and limitations of the PN in maintaining proteome integrity in the face of proteotoxic stresses, such as aggregate formation in neurodegenerative diseases. Finally, we outline various pharmacological interventions to ameliorate proteostasis imbalance.
Topics: Animals; Cell Differentiation; Homeostasis; Humans; Molecular Chaperones; Neurodegenerative Diseases; Protein Conformation; Protein Denaturation; Protein Folding; Proteins; Proteome; Proteostasis; Thermodynamics
PubMed: 30833457
DOI: 10.1101/cshperspect.a033951 -
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 -
The Journal of Physical Chemistry. B Apr 2023Protein stability is important in many areas of life sciences. Thermal protein unfolding is investigated extensively with various spectroscopic techniques. The...
Protein stability is important in many areas of life sciences. Thermal protein unfolding is investigated extensively with various spectroscopic techniques. The extraction of thermodynamic properties from these measurements requires the application of models. Differential scanning calorimetry (DSC) is less common, but is unique as it measures directly a thermodynamic property, that is, the heat capacity (). The analysis of () is usually performed with the chemical equilibrium two-state model. This is not necessary and leads to incorrect thermodynamic consequences. Here we demonstrate a straightforward model-independent evaluation of heat capacity experiments in terms of protein unfolding enthalpy Δ(), entropy Δ(), and free energy Δ()). This now allows the comparison of the experimental thermodynamic data with the predictions of different models. We critically examined the standard chemical equilibrium two-state model, which predicts a positive free energy for the native protein, and diverges distinctly from the experimental temperature profiles. We propose two new models which are equally applicable to spectroscopy and calorimetry. The Θ()-weighted chemical equilibrium model and the statistical-mechanical two-state model provide excellent fits of the experimental data. They predict sigmoidal temperature profiles for enthalpy and entropy, and a trapezoidal temperature profile for the free energy. This is illustrated with experimental examples for heat and cold denaturation of lysozyme and β-lactoglobulin. We then show that the free energy is not a good criterion to judge protein stability. More useful parameters are discussed, including protein cooperativity. The new parameters are embedded in a well-defined thermodynamic context and are amenable to molecular dynamics calculations.
Topics: Hot Temperature; Protein Denaturation; Proteins; Thermodynamics; Cold Temperature; Protein Unfolding; Calorimetry, Differential Scanning; Protein Folding
PubMed: 37040567
DOI: 10.1021/acs.jpcb.3c00882 -
Quarterly Reviews of Biophysics Feb 2020Proteins are molecular machines whose function depends on their ability to achieve complex folds with precisely defined structural and dynamic properties. The rational... (Review)
Review
Proteins are molecular machines whose function depends on their ability to achieve complex folds with precisely defined structural and dynamic properties. The rational design of proteins from first-principles, or de novo, was once considered to be impossible, but today proteins with a variety of folds and functions have been realized. We review the evolution of the field from its earliest days, placing particular emphasis on how this endeavor has illuminated our understanding of the principles underlying the folding and function of natural proteins, and is informing the design of macromolecules with unprecedented structures and properties. An initial set of milestones in de novo protein design focused on the construction of sequences that folded in water and membranes to adopt folded conformations. The first proteins were designed from first-principles using very simple physical models. As computers became more powerful, the use of the rotamer approximation allowed one to discover amino acid sequences that stabilize the desired fold. As the crystallographic database of protein structures expanded in subsequent years, it became possible to construct proteins by assembling short backbone fragments that frequently recur in Nature. The second set of milestones in de novo design involves the discovery of complex functions. Proteins have been designed to bind a variety of metals, porphyrins, and other cofactors. The design of proteins that catalyze hydrolysis and oxygen-dependent reactions has progressed significantly. However, de novo design of catalysts for energetically demanding reactions, or even proteins that bind with high affinity and specificity to highly functionalized complex polar molecules remains an importnant challenge that is now being achieved. Finally, the protein design contributed significantly to our understanding of membrane protein folding and transport of ions across membranes. The area of membrane protein design, or more generally of biomimetic polymers that function in mixed or non-aqueous environments, is now becoming increasingly possible.
Topics: Amino Acid Motifs; Animals; Binding Sites; Biotechnology; Catalysis; Crystallography, X-Ray; Humans; Hydrogen Bonding; Ions; Kinetics; Ligands; Macromolecular Substances; Protein Binding; Protein Denaturation; Protein Engineering; Protein Folding; Proteins; Zinc
PubMed: 32041676
DOI: 10.1017/S0033583519000131 -
Biophysical Reports Mar 2022Testing and predicting protein stability gained importance because proteins, including antibodies, became pharmacologically relevant in viral and cancer therapies....
Testing and predicting protein stability gained importance because proteins, including antibodies, became pharmacologically relevant in viral and cancer therapies. Isothermal scanning calorimetry is the principle method to study protein stability. Here, we use the excellent experimental heat capacity C(T) data from the literature for a critical inspection of protein unfolding as well as for the test of a new cooperative model. In the relevant literature, experimental temperature profiles of enthalpy, H(T), entropy, S(T), and free energy, G(T) are missing. First, we therefore calculate the experimental H(T), S(T), and G(T) from published C(T) thermograms. Considering only the unfolding transition proper, the heat capacity and all thermodynamic functions are zero in the region of the native protein. In particular, the free energy of the folded proteins is also zero and G(T) displays a trapezoidal temperature profile when cold denaturation is included. Second, we simulate the DSC-measured thermodynamic properties with a new molecular model based on statistical-mechanical thermodynamics. The model quantifies the protein cooperativity and predicts the aggregate thermodynamic variables of the system with molecular parameters only. The new model provides a perfect simulation of all thermodynamic properties, including the observed trapezoidal G(T) temperature profile. Importantly, the new cooperative model can be applied to a broad range of protein sizes, including antibodies. It predicts not only heat and cold denaturation but also provides estimates of the unfolding kinetics and allows a comparison with molecular dynamics calculations and quasielastic neutron scattering experiments.
PubMed: 36425081
DOI: 10.1016/j.bpr.2021.100037 -
Proceedings of the National Academy of... Jun 2021The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the...
The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the proteasome transitions from a substrate-accepting conformation (s1) to a set of substrate-processing conformations (s3 like), each stabilized by different intramolecular contacts. Tools to study these conformational changes remain limited, and although several interactions have been proposed to be important for stabilizing the proteasome's various conformations, it has been difficult to test these directly under equilibrium conditions. Here, we describe a conformationally sensitive Förster resonance energy transfer assay, in which fluorescent proteins are fused to Sem1 and Rpn6, which are nearer each other in substrate-processing conformations than in the substrate-accepting conformation. Using this assay, we find that two sets of interactions, one involving Rpn5 and another involving Rpn2, are both important for stabilizing substrate-processing conformations. Mutations that disrupt these interactions both destabilize substrate-processing conformations relative to the substrate-accepting conformation and diminish the proteasome's ability to successfully unfold and degrade hard-to-unfold substrates, providing a link between the proteasome's conformational state and its unfolding ability.
Topics: Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; Models, Molecular; Mutation; Proteasome Endopeptidase Complex; Protein Conformation; Protein Unfolding
PubMed: 34161281
DOI: 10.1073/pnas.2101004118