Changes in molar volume and heat capacity of actin upon polymerization.
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ABSTRACT:
SUBMITTER: Quirion F
PROVIDER: S-EPMC1134611 | biostudies-other | 1993 Nov
REPOSITORIES: biostudies-other
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Project description:In this paper we address the question of whether the burial of polar and nonpolar groups in the protein locale is indeed accompanied by the heat capacity changes, DeltaC(p), that have an opposite sign, negative for nonpolar groups and positive for polar groups. To accomplish this, we introduced amino acid substitutions at four fully buried positions of the ubiquitin molecule (Val5, Val17, Leu67, and Gln41). We substituted Val at positions 5 and 17 and Leu at position 67 with a polar residue, Asn. As a control, Ala was introduced at the same three positions. We also replaced the buried polar Gln41 with Val and Leu, nonpolar residues that have similar size and shape as Gln. As a control, Asn was introduced at Gln41 as well. The effects of these amino acid substitutions on the stability, and in particular, on the heat capacity change upon unfolding were measured using differential scanning calorimetry. The effect of the amino acid substitutions on the structure was also evaluated by comparing the (1)H-(15)N HSQC spectra of the ubiquitin variants. It was found that the Ala substitutions did not have a considerable effect on the heat capacity change upon unfolding. However, the substitutions of aliphatic side chains (Val or Leu) with a polar residue (Asn) lead to a significant (> 30%) decrease in the heat capacity change upon unfolding. The decrease in heat capacity changes does not appear to be the result of significant structural perturbations as seen from the HSQC spectra of the variants. The substitution of a buried polar residue (Gln41) to a nonpolar residue (Leu or Val) leads to a significant (> 25%) increase in heat capacity change upon unfolding. These results indicate that indeed the heat capacity change of burial of polar and nonpolar groups has an opposite sign. However, the observed changes in DeltaC(p) are several times larger than those predicted, based on the changes in water accessible surface area upon substitution.
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Project description:Whereas heat capacity changes (DeltaCPs) associated with folding transitions are commonplace in the literature of protein folding, they have long been considered a minor energetic contributor in nucleic acid folding. Recent advances in the understanding of nucleic acid folding and improved technology for measuring the energetics of folding transitions have allowed a greater experimental window for measuring these effects. We present in this review a survey of current literature that confronts the issue of DeltaCPs associated with nucleic acid folding transitions. This work helps to gather the molecular insights that can be gleaned from analysis of DeltaCPs and points toward the challenges that will need to be overcome if the energetic contribution of DeltaCP terms are to be put to use in improving free energy calculations for nucleic acid structure prediction.
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Project description:As a further elaboration of the recently devised Q10 scanning analysis ("Exceptionally high thermal sensitivity of rattlesnake TRPA1 correlates with peak current amplitude" [1]), the interval between current data points at two temperatures was shortened and the resulting parameters representing thermal sensitivities such as peak Q10s and temperature points of major thermosensitivity events are presented for two TRPA1 orthologues from rattlesnakes and boas. In addition, the slope factors from Boltzmann fitting and the change of molar heat capacity of temperature-evoked currents were evaluated and compared as alternative ways of thermal sensitivity appraisal of TRPA1 orthologues.
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Project description:This study examines the properties of a 4 × 2 matrix of aqueous cations and anions at concentrations up to 8.0 M. The apparent molar water volume, as calculated by subtracting the mass and volume of the ions from the corresponding solution density, was found to exceed the molar volume of ice in many concentrated electrolyte solutions, underscoring the nonideal behavior of these systems. The solvent properties of water were also analyzed by measuring the solubility of diketopiperazine (DKP) in 2.000 M salt solutions prepared from the same ion combinations. Solution rankings for DKP solubility were found to parallel the Hofmeister series for both cations and anions, whereas molar water volume concurred with the cation series only. The results are discussed within the framework of a desolvation energy model that attributes solute-specific changes in equilibria to solute-dependent changes in the free energy of bulk water.
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Project description:The heat capacity change, ?Cp, accompanying the folding/unfolding of macromolecules reflects their changing state of hydration. Thermal denaturation of the DNA duplex is characterized by an increase in ?Cp but of much lower magnitude than observed for proteins. To understand this difference, the changes in solvent accessible surface area (?ASA) have been determined for unfolding the B-form DNA duplex into disordered single strands. These showed that the polar component represents?~?55% of the total increase in ASA, in contrast to globular proteins of similar molecular weight for which the polar component is only about 1/3rd of the total. As the exposure of polar surface results in a decrease of ?Cp, this explains the much reduced heat capacity increase observed for DNA and emphasizes the enhanced role of polar interactions in maintaining duplex structure. Appreciation of a non-zero ?Cp for DNA has important consequences for the calculation of duplex melting temperatures (Tm). A modified approach to Tm prediction is required and comparison is made of current methods with an alternative protocol.
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Project description:Heat capacity changes are emerging as essential for explaining the temperature dependence of enzyme-catalysed reaction rates. This has important implications for enzyme kinetics, thermoadaptation and evolution, but the physical basis of these heat capacity changes is unknown. Here we show by a combination of experiment and simulation, for two quite distinct enzymes (dimeric ketosteroid isomerase and monomeric alpha-glucosidase), that the activation heat capacity change for the catalysed reaction can be predicted through atomistic molecular dynamics simulations. The simulations reveal subtle and surprising underlying dynamical changes: tightening of loops around the active site is observed, along with changes in energetic fluctuations across the whole enzyme including important contributions from oligomeric neighbours and domains distal to the active site. This has general implications for understanding enzyme catalysis and demonstrating a direct connection between functionally important microscopic dynamics and macroscopically measurable quantities.
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Project description:It has been suggested that heat capacity changes in enzyme catalysis may be the underlying reason for temperature optima that are not related to unfolding of the enzyme. If this were to be a common phenomenon, it would have major implications for our interpretation of enzyme kinetics. In most cases, the support for the possible existence of a nonzero (negative) activation heat capacity, however, only relies on fitting such a kinetic model to experimental data. It is therefore of fundamental interest to try to use computer simulations to address this issue. One way is simply to calculate the temperature dependence of the activation free energy and determine whether the relationship is linear or not. An alternative approach is to calculate the absolute heat capacities of the reactant and transition states from plain molecular dynamics simulations using either the temperature derivative or fluctuation formula for the enthalpy. Here, we examine these different approaches for a designer enzyme with a temperature optimum that is not caused by unfolding. Benchmark calculations for the heat capacity of liquid water are first carried out using different thermostats. It is shown that the derivative formula for the heat capacity is generally the most robust and insensitive to the thermostat used and its parameters. The enzyme calculations using this method give results in agreement with direct calculations of activation free energies and show no sign of a negative activation heat capacity. We also provide a simple scheme for the calculation of binding heat capacity changes, which is of clear interest in ligand design, and demonstrate it for substrate binding to the designer enzyme. Neither in that case do the simulations predict any negative heat capacity change.
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Project description:Heat shock (HS) initiates rapid, extensive, and evolutionarily conserved changes in transcription that are accompanied by chromatin decondensation and nucleosome loss at HS loci. Here we have employed in situ Hi-C to determine how heat stress affects long-range chromatin conformation in human and Drosophila cells. We found that compartments and topologically associating domains (TADs) remain unchanged by an acute HS. Knockdown of Heat Shock Factor 1 (HSF1), the master transcriptional regulator of the HS response, identified HSF1-dependent genes and revealed that up-regulation is often mediated by distal HSF1 bound enhancers. HSF1-dependent genes were usually found in the same TAD as the nearest HSF1 binding site. Although most interactions between HSF1 binding sites and target promoters were established in the nonheat shock (NHS) condition, a subset increased contact frequency following HS. Integrating information about HSF1 binding strength, RNA polymerase abundance at the HSF1 bound sites (putative enhancers), and contact frequency with a target promoter accurately predicted which up-regulated genes were direct targets of HSF1 during HS. Our results suggest that the chromatin conformation necessary for a robust HS response is preestablished in NHS cells of diverse metazoan species.
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Project description:Actin polymerization is a universal mechanism to drive plasma membrane protrusion in motile cells. One apparent exception to this rule is continuing, or even accelerated outgrowth of neuronal processes in the presence of actin polymerization inhibitors. This fact together with a key role of microtubule dynamics in neurite outgrowth led to the concept that microtubules directly drive plasma membrane protrusion, either in the course of polymerization or motor-driven sliding. Surprisingly, a possibility that unextinguished actin polymerization drives neurite outgrowth in the presence of actin drugs was not explored. We show that cultured hippocampal neurons treated with cytochalasin D or latrunculin B contained dense accumulations of branched actin filaments at ?50% of neurite tips at all tested drug concentrations (1-10 ?M). Actin polymerization was required for neurite outgrowth, because only low concentrations of either inhibitor increased the length and/or a number of neurites, whereas high concentrations inhibited neurite outgrowth. Importantly, neurites undergoing active elongation invariably contained a bright F-actin patch at the tip, whereas actin-depleted neurites never elongated, even though they still contained dynamic microtubules. Stabilization of microtubules by taxol treatment did not stop elongation of cytochalasin d-treated neurites. We conclude that actin polymerization is indispensable for neurite elongation.
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Project description:The 25-kD inhibitor of actin polymerization (25-kD IAP), isolated from turkey smooth muscle (Miron, T., M. Wilchek, and B. Geiger, 1988. Eur. J. Biochem. 178:543-553), is shown here to be a low molecular mass heat shock protein (HSP). Direct sequence analysis of the purified protein, as well as cloning and sequencing of the respective cDNA, disclosed a high degree of homology (67% identity, 80% similarity) to the human 27-kD HSP. Southern blot of chicken genomic DNA disclosed one band, suggesting the presence of a single gene, and Northern blot analysis revealed abundant transcript of approximately 1 kb in gizzard and heart tissues and lower amounts in total 18-d chick embryo RNA and in cultured fibroblasts. Exposure of the latter cells to 45 degrees C resulted in over 15-fold increase in the apparent level of the 25-kD IAP protein, confirming that its expression is regulated by heat shock. Immunofluorescent microscopic localization indicated that after heat treatment, the levels of the 25-kD IAP were markedly increased and the protein was apparently associated with cytoplasmic granules. Heat shock also had a transient, yet prominent, effect on the microfilament system in cultured fibroblasts: stress fibers disintegrated within 10-15 min after incubation at 45 degrees C, yet upon further incubation at the elevated temperature, conspicuous actin bundles were apparently reformed.
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