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Biochemistry Jun 2023Caspases are evolutionarily conserved cysteinyl proteases that are integral in cell development and apoptosis. All apoptotic caspases evolved from a common ancestor into...
Caspases are evolutionarily conserved cysteinyl proteases that are integral in cell development and apoptosis. All apoptotic caspases evolved from a common ancestor into two distinct subfamilies with either monomeric (initiators) or dimeric (effectors) oligomeric states. The regulation of apoptosis is influenced by the activation mechanism of the two subfamilies, but the evolution of the well-conserved caspase-hemoglobinase fold into the two subfamilies is not well understood. We examined the folding landscape of monomeric caspases from two coral species over a broad pH range of 3-10.5. On an evolutionary timescale, the two coral caspases diverged from each other approximately 300 million years ago, and they diverged from human caspases about 600 million years ago. Our results indicate that both proteins have overall high stability, ∼15 kcal mol, near the physiological pH range (pH 6-8) and unfold via two partially folded intermediates, I and I, that are in equilibrium with the native and the unfolded state. Like the dimeric caspases, the monomeric coral caspases undergo a pH-dependent conformational change resulting from the titration of an evolutionarily conserved site. Data from molecular dynamics simulations paired with limited proteolysis and MALDI-TOF mass spectrometry show that the small subunit of the monomeric caspases is unstable and unfolds prior to the large subunit. Overall, the data suggest that all caspases share a conserved folding landscape, that a conserved allosteric site can be fine-tuned for species-specific regulation, and that the subfamily of stable dimers may have evolved to stabilize the small subunit.
Topics: Humans; Protein Folding; Caspases; Protein Denaturation
PubMed: 37337671
DOI: 10.1021/acs.biochem.3c00004 -
Biochemistry Mar 2022The high concentration of macromolecules in cells affects the stability of proteins and protein complexes via hard repulsions and chemical interactions, yet few studies...
The high concentration of macromolecules in cells affects the stability of proteins and protein complexes via hard repulsions and chemical interactions, yet few studies have focused on chemical interactions. We characterized the domain-swapped dimer of the B1 domain of protein G in buffer and cells by using heteronuclear, multidimensional nuclear magnetic resonance spectroscopy. In buffer, the monomer is a partially folded molten globule, but that species is not observed in cells. Experiments using urea suggest that the monomer is unfolded in cells, but again, the molten-globule form of the monomer is absent. The data suggest that attractive chemical interactions in the cytoplasm unfold the molten globule. We conclude that the intracellular environment not only modulates the stability of protein complexes but also can change the species present, reinforcing the idea that chemical interactions are more important than hard repulsions in cells.
Topics: Circular Dichroism; Macromolecular Substances; Nuclear Magnetic Resonance, Biomolecular; Polymers; Protein Conformation; Protein Denaturation; Protein Folding; Proteins; Urea
PubMed: 35188746
DOI: 10.1021/acs.biochem.1c00780 -
Protein and Peptide Letters 2020Use of organic molecules as co-solvent with water, the ubiquitous biological solvent, to perturb the structure of proteins is popular in the research area of protein... (Review)
Review
Use of organic molecules as co-solvent with water, the ubiquitous biological solvent, to perturb the structure of proteins is popular in the research area of protein structure and folding. These organic co-solvents are believed to somehow mimic the environment near the cell membrane. Apart from that they induce non-native states which can be present in the protein folding pathway or those states also may be representative of the off pathway structures leading to amyloid formation, responsible for various fatal diseases. In this review, we shall focus on organic co-solvent induced structure perturbation of various members of lectin family. Lectins are excellent model systems for protein folding study because of its wide occurrence, diverse structure and versatile biological functions. Lectins were mainly perturbed by two fluoroalcohols - 2,2,2- trifluoroethanol and 1,1,1,3,3,3-hexafluoroisopropanol whereas glycerol, ethylene glycol and polyethylene glycols were used in some cases. Overall, all native lectins were denatured by alcohols and most of the denatured lectins have predominant helical secondary structure. But characterization of the helical states and the transition pathway for various lectins revealed diverse result.
Topics: Alcohols; Circular Dichroism; Lectins; Models, Molecular; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Solvents; Water
PubMed: 31682206
DOI: 10.2174/0929866526666191104145511 -
Physical Chemistry Chemical Physics :... Mar 2022Experimental measurements of the thermal effects of the same osmolytes on two different globular proteins, C-reactive protein (CRP) and tumor necrosis factor alpha...
Experimental measurements of the thermal effects of the same osmolytes on two different globular proteins, C-reactive protein (CRP) and tumor necrosis factor alpha (TNFα), have shown that quantifying the change in the denaturing temperature leads to some results that are unique to each protein. In order to find osmolyte-dependent parameters that can be applied more consistently from protein to protein, this work considers, instead, the overall free energy change associated with that denaturation using coarse-grained models. This is enabled by using theoretical fluid equations that take into account the exclusion of water and osmolyte from the volume occupied by the protein in both its native and denatured forms. Assuming ideal geometric models of the two protein states whose sizes are based on the protein's surface area in each form, and taking into account the density of the aqueous osmolyte solution, the free energy change due to the change in geometry can be calculated. The overall change in free energy of the system is found from that quantity and other protein- and osmolyte-specific parameters, which are determined using the experimental concentration and temperature results. We find that these fitted parameters accurately reproduce experimental results and also show consistent patterns from protein to protein. We also consider two different model geometries of the denatured protein and find little impact on the use of one or the other. Defining the effects of the osmolyte in terms of free energy also allows for prediction of overall phase change behavior, including cold denaturation.
Topics: Osmolar Concentration; Protein Denaturation; Proteins; Temperature; Thermodynamics
PubMed: 35169823
DOI: 10.1039/d1cp04460e -
Biophysical Journal Jul 2023The actin filament network is in part remodeled by the action of a family of filament severing proteins that are responsible for modulating the ratio between monomeric...
The actin filament network is in part remodeled by the action of a family of filament severing proteins that are responsible for modulating the ratio between monomeric and filamentous actin. Recent work on the protein actophorin from the amoeba Acanthamoeba castellani identified a series of site-directed mutations that increase the thermal stability of the protein by 22°C. Here, we expand this observation by showing that the mutant protein is also significantly stable to both equilibrium and kinetic chemical denaturation, and employ computer simulations to account for the increase in thermal or chemical stability through an accounting of atomic-level interactions. Specifically, the potential of mean force (PMF) can be obtained from steered molecular dynamics (SMD) simulations in which a protein is unfolded. However, SMD can be inefficient for large proteins as they require large solvent boxes, and computationally expensive as they require increasingly many SMD trajectories to converge the PMF. Adaptive steered molecular dynamics (ASMD) overcomes the second of these limitations by steering the particle in stages, which allows for convergence of the PMF using fewer trajectories compared with SMD. Use of the telescoping water scheme within ASMD partially overcomes the first of these limitations by reducing the number of waters at each stage to only those needed to solvate the structure within a given stage. In the PMFs obtained from ASMD, the work of unfolding Acto-2 was found to be higher than the Acto-WT by approximately 120 kCal/mol and reflects the increased stability seen in the chemical denaturation experiments. The evolution of the average number of hydrogen bonds and number of salt bridges during the pulling process provides a mechanistic view of the structural changes of the actophorin protein as it is unfolded, and how it is affected by the mutation in concert with the energetics reported through the PMF.
Topics: Acanthamoeba; Actins; Amoeba; Molecular Dynamics Simulation; Solvents; Protein Denaturation
PubMed: 36461639
DOI: 10.1016/j.bpj.2022.11.2941 -
International Journal of Molecular... Apr 2023Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a...
Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures ( 67 °C) but undergoes significant unfolding at room temperature due to cold denaturation. Little is known about the cold denaturation of thermophilic proteins, although it can significantly limit their applications. We investigated the cold denaturation of thermophilic multidomain protein translation initiation factor 2 (IF2) from . IF2 is a GTPase that binds to ribosomal subunits and initiator fMet-tRNA during the initiation of protein biosynthesis. In the presence of 9 M urea, measurements in the far-UV region by circular dichroism were used to capture details about the secondary structure of full-length IF2 protein and its domains during cold and hot denaturation. Cold denaturation can be suppressed by salt, depending on the type, due to the decreased heat capacity. Thermodynamic analysis and mathematical modeling of the denaturation process showed that salts reduce the cooperativity of denaturation of the IF2 domains, which might be associated with the high frustration between domains. This characteristic of high interdomain frustration may be the key to satisfying numerous diverse contacts with ribosomal subunits, translation factors, and tRNA.
Topics: Prokaryotic Initiation Factor-2; Cold Temperature; Protein Biosynthesis; Thermodynamics; Hot Temperature; Sodium Chloride; Sodium Chloride, Dietary; Protein Denaturation
PubMed: 37047761
DOI: 10.3390/ijms24076787 -
Quarterly Reviews of Biophysics Jun 2020Darwin's theory of evolution emphasized that positive selection of functional proficiency provides the fitness that ultimately determines the structure of life, a view... (Review)
Review
Darwin's theory of evolution emphasized that positive selection of functional proficiency provides the fitness that ultimately determines the structure of life, a view that has dominated biochemical thinking of enzymes as perfectly optimized for their specific functions. The 20th-century modern synthesis, structural biology, and the central dogma explained the machinery of evolution, and nearly neutral theory explained how selection competes with random fixation dynamics that produce molecular clocks essential e.g. for dating evolutionary histories. However, quantitative proteomics revealed that selection pressures not relating to optimal function play much larger roles than previously thought, acting perhaps most importantly via protein expression levels. This paper first summarizes recent progress in the 21st century toward recovering this universal selection pressure. Then, the paper argues that proteome cost minimization is the dominant, underlying 'non-function' selection pressure controlling most of the evolution of already functionally adapted living systems. A theory of proteome cost minimization is described and argued to have consequences for understanding evolutionary trade-offs, aging, cancer, and neurodegenerative protein-misfolding diseases.
Topics: Adenosine Triphosphate; Amino Acids; Animals; Biological Evolution; Computational Biology; Humans; Kinetics; Molecular Conformation; Protein Denaturation; Protein Folding; Proteome; Proteomics; Selection, Genetic; Solvents; Temperature
PubMed: 32624048
DOI: 10.1017/S0033583520000037 -
The Journal of Physical Chemistry. B May 2021Proteins are stable over a narrow temperature range, with hot and cold denaturation occurring outside of this window, both of which adversely affect protein function....
Proteins are stable over a narrow temperature range, with hot and cold denaturation occurring outside of this window, both of which adversely affect protein function. While hot unfolding is entropically driven, cold denaturation, on the other hand, results from a more favorable free energy associated with the interaction of water with apolar groups at low temperature. Because of the key role of water in this latter process, capturing cold denaturation using implicit solvent models is challenging. We propose here a novel computational approach to develop an implicit solvent model that accounts for both hot and cold denaturation in simulations involving atomistically detailed protein representations. By mining a large number of protein structures solved by nuclear magnetic resonance, we derive transfer free energy contributions for the backbone and amino acids side chains representing the transfer of these moieties between water at two different temperatures. Using Trp-cage as a model system, we show that the implicit solvent model constructed using these temperature-dependent free energies of transfer recovers the parabolic temperature dependence of protein stability, capturing both hot and cold denaturation. The resulting cold-unfolded conformations show reduced secondary structure content but preserve most of their internal hydrogen-bonding network, in contrast to the extended configurations with no hydrogen-bonding populated during heat-induced denaturation.
Topics: Cold Temperature; Entropy; Hot Temperature; Hydrogen Bonding; Protein Denaturation; Protein Folding; Solvents; Thermodynamics; Water
PubMed: 33988995
DOI: 10.1021/acs.jpcb.1c01694 -
Proceedings of the National Academy of... Aug 2021The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift...
The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute-protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute-protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, [Formula: see text], and an effective correlation time, τ The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute-protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the [Formula: see text] values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state.
Topics: Animals; Drosophila Proteins; Drosophila melanogaster; Models, Molecular; Protein Denaturation; Protein Folding; Protein Unfolding; Solvents; Thermodynamics; src Homology Domains
PubMed: 34404723
DOI: 10.1073/pnas.2112021118 -
Molecules (Basel, Switzerland) Nov 2023MDM2 is an E3 ubiquitin ligase which is crucial for the degradation and inhibition of the key tumor-suppressor protein p53. In this work, we explored the stability and...
MDM2 is an E3 ubiquitin ligase which is crucial for the degradation and inhibition of the key tumor-suppressor protein p53. In this work, we explored the stability and the conformational features of the N-terminal region of MDM2 (N-MDM2), through which it binds to the p53 protein as well as other protein partners. The isolated domain possessed a native-like conformational stability in a narrow pH range (7.0 to 10.0), as shown by intrinsic and 8-anilinonapthalene-1-sulfonic acid (ANS) fluorescence, far-UV circular dichroism (CD), and size exclusion chromatography (SEC). Guanidinium chloride (GdmCl) denaturation followed by intrinsic and ANS fluorescence, far-UV CD and SEC at physiological pH, and differential scanning calorimetry (DSC) and thermo-fluorescence experiments showed that (i) the conformational stability of isolated N-MDM2 was very low; and (ii) unfolding occurred through the presence of several intermediates. The presence of a hierarchy in the unfolding intermediates was also evidenced through DSC and by simulating the unfolding process with the help of computational techniques based on constraint network analysis (CNA). We propose that the low stability of this protein is related to its inherent flexibility and its ability to interact with several molecular partners through different routes.
Topics: Protein Folding; Tumor Suppressor Protein p53; Protein Denaturation; Protein Conformation; Circular Dichroism; Hydrogen-Ion Concentration; Spectrometry, Fluorescence; Calorimetry, Differential Scanning
PubMed: 38005300
DOI: 10.3390/molecules28227578