Fluorescence study of the high pressure‐induced denaturation of skeletal muscle actin

Abstract

] made a thorough study of the e ect of pressure on G-and F-actins. However, all of the measurements in their study were made after the release of pressure. In the present experiment in situ observations were attempted by using eATP to obtain further detailed kinetic and thermodynamic information about the behaviour of actin under pressure. The dissociation rate constants of nucleotides from actin molecules (the decay curve of the intensity of¯uorescence of eATP-G-actin or eADP±F-actin) followed ®rst-order kinetics. The volume changes for the denaturation of G-actin and F-actin were estimated to be )72 mLámol )1 and )67 mLámol )1 in the presence of ATP, respectively. Changes in the intensity of¯uorescence of F-actin whilst under pressure suggested that eADP±F-actin was initially depolymerized to eADP±G-actin; subsequently there was quick exchange of the eADP for free eATP, and then polymerization occurred again with the liberation of phosphate from eATP bound to G-actin in the presence of excess ATP. In the higher pressure range (> 250 MPa), the partial collapse of the three-dimensional structure of actin, which had been depolymerized under pressure, proceeded immediately after release of the nucleotide, so that it lost the ability to exchange bound ADP with external free ATP and so was denatured irreversibly. An experiment monitoring eATP¯uorescence also demonstrated that, in the absence of Mg 2+ -ATP, the dissociation of 1 actin-heavy meromyosin (HMM) complex into actin and HMM did not occur under high pressure.

Polymeric Structures and Dynamic Properties of the Bacterial Actin AlfA

“…Exerting pressure on a filamentous structure can lead to destruction of noncovalent bonds, loss of nucleotide, and, subsequently, filament depolymerization, as shown for actin and ParM filaments. 11,17 AlfA filaments disintegrated almost linearly with rising pressure, with complete disintegration reached at about 1700 bar (Fig. S4b and c).…”

“…5d). The behavior of AlfA filaments under pressure differed both from that of F-actin, which was a stable filament until about 2000 bar, 17 and from that of ParM, which dissociated rapidly at low pressures around 400-500 bar. 11…”

Combined Effects of Temperature, Pressure, and Co‐Solvents on the Polymerization Kinetics of Actin

“…[10] Moreover,inv itro studies have shown that pressure shifts the equilibrium of the monomer-to-filament transformation of actin towards the monomeric state of the protein, and this equilibrium shift has been found to be species-dependent. [11][12][13][14][15] In this study,w es et out to quantitativelye xplore the kinetics of the polymerization reactiono fa ctin, focusing on the early events, as af unction of temperature and pressure to gain am echanistic understanding of their limitations on actin assembly.H igh-pressure stopped-flow measurements at selected temperatures enabled us to determine the activationv olume, DV°,o ft he process, whichi nt urn sheds light on the inhibitory process of pressure. Moreover,i nc ombination with quantitative Monte Carlo simulations of the polymerization process, we were able to elucidate which features and kinetic parameters are essential in determining their kinetic stability.…”

Filament Structure, Organization, and Dynamics in MreB Sheets

“…Exerting pressure on a filamentous structure, can lead to the destruction of noncovalent interactions, loss of nucleotide and subsequently to filament depolymerization, as shown for actin and ParM filaments (24,8). MreB filaments were stable unto about 2000 bar after which they disintegrated (Fig.…”