The use of hydrostatic pressure as a tool to study viruses and other macromolecular assemblages

Silva, Jerson L.; Foguel, Débora; Poian, Andrea T. Da; Prevelige, Peter E.

doi:10.1016/s0959-440x(96)80071-6

Cited by 135 publications

(137 citation statements)

References 74 publications

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“…A shift of 157 Ϯ 9 bar was measured in the pressure that causes 50% dissociation (p 1/2 ) (Fig. 2B), thus showing a concentration dependence characteristic of a dimer-monomer dissociation equilibrium (33)(34)(35). The measured ⌬p 1/2 is equal to the theoretical value determined from Equation 4 ( Table I).…”

Section: Resultsmentioning

confidence: 55%

“…This equation permits the calculation of the standard volume change ⌬V, from measurements at a fixed protein concentration C, at different pressures (33)(34)(35). A change in protein concentration from C 1 to C 2 at a fixed pressure results in a parallel displacement ⌬p of the plot of ln[(␣ p ) 2 /(1 Ϫ ␣ p )] versus p. At 50% dissociation, this shift (⌬p 1/2 ) in pressure upon change in concentration is given by (33) the following equation.…”

Section: Methodsmentioning

confidence: 99%

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LexA Repressor Forms Stable Dimers in Solution

Mohana‐Borges

Pacheco

Sousa

et al. 2000

Journal of Biological Chemistry

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Cooperativity in the interactions among proteins subunits and DNA is crucial for DNA recognition. LexA repressor was originally thought to bind DNA as a monomer, with cooperativity leading to tighter binding of the second monomer. The main support for this model was a high value of the dissociation constant for the LexA dimer (micromolar range). Here we show that the protein is a dimer at nanomolar concentrations under different conditions. The reversible dissociation of LexA dimer was investigated by the effects of hydrostatic pressure or urea, using fluorescence emission and polarization to monitor the dissociation process. The dissociation constant lies in the picomolar range (lower than 20 pM). LexA monomers associate with an unusual large volume change (340 ml/mol), indicating the burial of a large surface area upon dimerization. Whereas nonspecific DNA has no stabilizing effect, specific DNA induces tightening of the dimer and a 750-fold decrease in the K d . In contrast to the previous model, a tight dimer rather than a monomer is the functional repressor. Accordingly, the LexA dimer only loses its ability to recognize a specific DNA sequence by RecA-induced autoproteolysis. Our work provides insights into the linkage between protein-protein interactions, DNA recognition, and DNA repair.DNA-binding proteins recognize specific sequences of DNA, and the molecular basis for the interactions is currently the focus of intense research (1-3). X-ray crystallography and nuclear magnetic resonance spectroscopy have revealed that the reading of a DNA sequence is achieved by a combination of van der Waals', electrostatic, and hydrogen bonds (2, 3). The cooperative interactions among protein subunits, regulatory proteins, enzymes, and DNA are responsible for many biological functions, such as transcription, DNA replication, DNA repair, and differentiation (2, 4 -8). Most sequence-specific DNA-binding proteins are dimers or tetramers, and in many cases, the crystal structure of the protein-DNA complex reveals that each subunit of a dimer contacts each half of the operator (2, 3). In several cases dimerization only occurs upon DNA binding, as for example with the DNA-binding domains of the steroidnuclear receptors (9). Dimerization upon binding to DNA was also shown to be the relevant pathway for some basic region leucine zipper and basic region helix-loop-helix zipper transcription factors (10). However, in many DNA-binding proteins the subunits associate with high affinity in solution in the absence of DNA, with K d values in the range of 10 Ϫ7 -10 Ϫ10 M (2, 3, 11-13). In these latter cases, a free energy linkage between folding/oligomerization and DNA binding is still crucial for the recognition process (14 -18). LexA repressor is a 202-residue protein that regulates transcription in Escherichia coli of the SOS system, with about 17 genes that encode proteins involved in DNA repair (19,20). LexA has two domains, and it undergoes autocleavage at residues 84 -85 in a flexible hinge between the two domains (21). The so...

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Section: Resultsmentioning

confidence: 55%

Section: Methodsmentioning

confidence: 99%

LexA Repressor Forms Stable Dimers in Solution

Mohana‐Borges

Pacheco

Sousa

et al. 2000

Journal of Biological Chemistry

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“…14 That entropy convergence coincides with the observed temperature convergence of the entropy of protein unfolding and thus provides a thermodynamic foundation for the hydrophobic-core model of protein folding. 6,7,11,12,16,17,18 In addition, we will suggest a molecular mechanism for pressure denaturation of proteins 19,20,21,22,23,24,25,26 and discuss its consequences on folding kinetics and the characteristics of the ensemble of unfolded protein structures. 15 Our explanation of pressure denaturation invokes experimental observations regarding the differences in the structures of heat and pressure-denatured proteins, the latter being more compact.…”

Section: Introductionmentioning

confidence: 99%

Hydrophobic Effects on a Molecular Scale

et al. 1998

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A theoretical approach is developed to quantify hydrophobic hydration and interactions on a molecular scale, with the goal of insight into the molecular origins of hydrophobic effects. The model is based on the fundamental relation between the probability for cavity formation in bulk water resulting from molecular-scale density fluctuations, and the hydration free energy of the simplest hydrophobic solutes, hard particles. This probability is estimated using an information theory (IT) approach, incorporating experimentally available properties of bulk water -the density and radial distribution function. The IT approach reproduces the simplest hydrophobic effects: hydration of spherical nonpolar solutes, the potential of mean force (PMF) between methane molecules, and solvent contributions to the torsional equilibrium of butane. Applications of this approach to study temperature and pressure effects provide new insights into the thermodynamics and kinetics of protein folding. The IT model relates the hydrophobic-entropy convergence observed in protein unfolding experiments to the macroscopic isothermal compressibility of water. A novel explanation for pressure denaturation of proteins follows from an analysis of the pressure stability of hydrophobic aggregates, suggesting that water penetrates the hydrophobic core of proteins at high pressures. This resolves a long-standing puzzle, whether pressure denaturation contradicts the hydrophobic-core model of protein stability. Finally, issues of "dewetting" of molecularly large nonpolar solutes are discussed in the context of a recently developed perturbation theory approach.

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“…The pressure denaturation of monomeric proteins, the dissociation of oligomers, and the effects of pressure on macromolecular assemblages have provided insights into the microscopic mechanism of protein folding and the role of solvent in this process (Zipp & Kauzmann, 1973;Li et al, 1976;Chryssomallis et al, 1981;Weber & Drickamer, 1983;Silva et al, 1986;Silva & Weber, 1993;Weber, 1993;Dufour et al, 1994;Peng et al, 1994;Schulte et al, 1995;Silva et al, 1996). These effects are often reversible but can show different degrees of hysteresis.…”

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confidence: 99%

Effects of pressure on the structure of metmyoglobin: Molecular dynamics predictions for pressure unfolding through a molten globule intermediate

et al. 1998

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We investigated the pathway for pressure unfolding of metmyoglobin using molecular dynamics (MD) for a range of pressures (0.1 MPa to 1.2 GPa) and a temperature of 300 K. We find that the unfolding of metmyoglobin proceeds via a two-step mechanism native + molten globule intermediate + unfolded, where the molten globule forms at 700 MPa. The simulation describes qualitatively the experimental behavior of metmyoglobin under pressure. We find that unfolding of the alpha-helices follows the sequence of migrating hydrogen bonds (i,i + 4) + (i,i + 2).Keywords: molecular dynamics; molten globule; myoglobin; pressure; unfolding The effect of pressure on chemical and biological systems can lead to improved understanding of the fundamental interactions and to improved processes. Thus, high pressure has been used in food sterilization, extraction procedures, bioconversion using barophilic microorganisms, and enzymology under supercritical conditions (Balny et al., 1992). In addition, microbiology under deep-sea high pressure conditions offers biotechnological opportunities and may provide insights into the origin of life. Microorganisms in the deep-sea live at high pressures (1 10 MPa at 10,660 m) at both low and high temperature and in darkness (Yayanos, 1995).Experimental techniques such as optical spectroscopy, Raman scattering, and NMR have been used to observe pressure effects on proteins (Weber & Drickamer, 1983;Frauenfelder et al., 1990;Jaenicke, 1991;Silva & Weber, 1993;Gross & Jaenicke, 1994 . The pressure denaturation of monomeric proteins, the dissociation of oligomers, and the effects of pressure on macromolecular assemblages have provided insights into the microscopic mechanism of protein folding and the role of solvent in this process (Zipp & Kauzmann, 1973;Li et al., 1976;Chryssomallis et al., 1981;Weber & Drickamer, 1983;Silva et al., 1986;Silva & Weber, 1993;Weber, 1993;Dufour et al., 1994;Peng et al., 1994;Schulte et al., 1995;Silva et al., 1996). These effects are often reversible but can show different degrees of hysteresis.Despite this progress, the detailed atomistic changes involved in pressure transformations have not been well characterized. To provide such information we carried out molecular dynamics (MD) simulations on metmyoglobin as a function of applied external pressure. We selected myoglobin since its folding mechanism and protein folding intermediates have been studied experimentally by many techniques. We find that the applied pressure perturbs the native structure of the protein, eventually destabilizing to denature the molecule. By examining the conformation of the intermediates from the MD, we determined the pathway for unfolding. The results are in agreement with the experiment. 2301

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The use of hydrostatic pressure as a tool to study viruses and other macromolecular assemblages

Cited by 135 publications

References 74 publications

LexA Repressor Forms Stable Dimers in Solution

LexA Repressor Forms Stable Dimers in Solution

Hydrophobic Effects on a Molecular Scale

Effects of pressure on the structure of metmyoglobin: Molecular dynamics predictions for pressure unfolding through a molten globule intermediate

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