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

Abstract

In vivo studies have shown that the cytoskeleton of cells is very sensitive to changes in temperature and pressure. In particular, actin filaments get depolymerized when pressure is increased up to several hundred bars, conditions that are easily encountered in the deep sea. We quantitatively evaluate the effects of temperature, pressure, and osmolytes on the kinetics of the polymerization reaction of actin by high-pressure stopped-flow experiments in combination with fluorescence detection and an integrative stochastic simulation of the polymerization process. We show that the compatible osmolyte trimethylamine-N-oxide is not only able to compensate for the strongly retarding effect of chaotropic agents, such as urea, on actin polymerization, it is also able to largely offset the deteriorating effect of pressure on actin polymerization, thereby allowing biological cells to better cope with extreme environmental conditions.

Kinetic Insights into the Elongation Reaction of Actin Filaments as a Function of Temperature, Pressure, and Macromolecular Crowding

“…[7,35] Owing to the sensitivity of pyrene to microenvironmental polarity change during filament formation,its fluorescenceintensity, I,increases proportionally with the concentration or length of F-actin. [23,38] The addition of gelsolin,a nd hence,i ncreased concentrationo f preformed GA 2 nuclei leads to the nucleation phase being by-passed, and thus to ap rogressively single exponential timecourse in ag elsolin concentration-dependentm anner,r eflecting filament elongation towardt he pointed end ( Figure 1B). [23,38] The addition of gelsolin,a nd hence,i ncreased concentrationo f preformed GA 2 nuclei leads to the nucleation phase being by-passed, and thus to ap rogressively single exponential timecourse in ag elsolin concentration-dependentm anner,r eflecting filament elongation towardt he pointed end ( Figure 1B).…”

“…[23,38] The addition of gelsolin,a nd hence,i ncreased concentrationo f preformed GA 2 nuclei leads to the nucleation phase being by-passed, and thus to ap rogressively single exponential timecourse in ag elsolin concentration-dependentm anner,r eflecting filament elongation towardt he pointed end ( Figure 1B). [38] As the slow de novo nucleation step is bypassed by the GA 2 complex, the impact of the less frequent spontaneous fragmentation of Factin on the elongation kinetics can be neglected (see the Supporting Information, Table S1). [38] As the slow de novo nucleation step is bypassed by the GA 2 complex, the impact of the less frequent spontaneous fragmentation of Factin on the elongation kinetics can be neglected (see the Supporting Information, Table S1).…”

Crowders and Cosolvents—Major Contributors to the Cellular Milieu and Efficient Means to Counteract Environmental Stresses

“…Not only protein folding, but also other biomolecular processes are markedly affected by such osmolytes. Recently, our laboratory could show that TMAO is able to significantly reduce the activation volume of the nucleation step for the self‐assembly reaction of actin, a central player of the cytoskeleton . This means that TMAO may act as a chemical chaperone to assist the rather pressure‐sensitive de novo formation of actin filaments in deep‐sea organisms.…”

“…Recently,o ur laboratory could show that TMAO is ablet os ignificantly reduce the activation volumeo fthen ucleations tep for the self-assembly reaction of actin, ac entral player of the cytoskeleton. [165] This means that TMAO may act as ac hemical chaperone to assist the rather pressure-sensitive de novo formation of actin filaments in deep-sea organisms. Furthermore, TMAO is able to reduce the sub-nanosecond dynamics of the protein's hydrogen atoms, presumably via water structure enhancement.…”

Combined pressure and cosolvent effects on enzyme activity – a high-pressure stopped-flow kinetic study on α-chymotrypsin

“…[33][34][35] The increase in optical absorbance at 410 nm due to formation of the product p-nitroaniline (with molar absorption coefficient e 410 = 8800 M À1 cm À1 ) was recorded using the built-in absorbance spectrometer of the systems. [33][34][35] The increase in optical absorbance at 410 nm due to formation of the product p-nitroaniline (with molar absorption coefficient e 410 = 8800 M À1 cm À1 ) was recorded using the built-in absorbance spectrometer of the systems.…”