Cosolvent and crowding effects on the polymerization kinetics of actin

Abstract

We studied the effects of kosmotropic and chaotropic cosolvents, trimethylamine-N-oxide (TMAO) and urea, as well as crowding agents (dextran) on the polymerization reaction of actin. Time-lapse fluorescence intensity and anisotropy experiments were carried out to yield information about the kinetics of the polymerization process. To also quantitatively describe the effects, cosolvents and crowding impose on the underlying rate constants of the G-to-F-transformation, an integrative stochastic simulation model was applied. Drastic and diverse changes in the lag phase and association rates as well as the critical actin concentration were observed under different solvent conditions. The association rate constant is drastically increased by TMAO but decreased by urea. In mixtures of these osmolytes, TMAO counteracts not only the deleterious effect of urea on protein structure and stability, but also on the protein-protein interactions in the course of actin polymerization. Owing to the excluded volume effect, cell-like macromolecular crowding conditions increase the nucleation and association rates by one order of magnitude. Our results clearly reveal the pronounced sensitivity of the actin polymerization reaction to changes in cosolvent conditions and the presence of macromolecular crowding, and suggest that such effects should be taken into account in any discussion of the actin polymerization reaction in vivo.

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).…”

“…They exhibit different association/dissociation rates, and thus different critical concentrations, leadingt ot he phenomenon of treadmilling. [17][18][19][20][21][22] It has been shownb yu sing synthetic crowding agents that the excluded volumee ffect accelerates actin nucleation [23] ande qualizes the critical concentrations of adenosine triphosphate (ATP)-actin and the less stable adenosine diphosphate (ADP)-actin. In par-ticular, the concentration of macromolecules in the cytosol can reach 400 gL À1 ,r esulting in ar eduction of the accessiblec ellular volume of up to 30-40 %, and thus often causing an entropic effect on folding and association equilibria of the biomolecules (termeda sexcluded volumee ffect).…”

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

“…Many crowding studies based on the use of artificial macromolecular agents such as Ficoll, dextran and PEG, reported on the ability of the excluded volume effect to stabilize proteins, stabilize RNA tertiary structure and increase ribozyme activity, enhance protein‐protein associations as well as promote protein polymerization and aggregation . For example, Erlkamp et al.…”

“…[188] Many crowding studies based on the use of artificial macromolecular agentss uch as Ficoll, dextran and PEG, reported on the ability of the excluded volume effect to stabilize proteins, [181,189,190] stabilize RNA tertiary structure and increase ribo-zyme activity, [191][192][193][194] enhance protein-protein associations [195] as well as promote protein polymerization and aggregation. [196][197][198][199][200][201][202][203] For example, Erlkamp et al applied FT-IR spectroscopic, smallangle X-ray scattering and calorimetric measurements to explore the effect of macromolecular crowding (30 wt %F icoll 70) on the temperature-and pressure-dependent stability diagram the protein SNase. [181] As shown in Figure 7b,t he temperature-and pressure-induced equilibrium unfolding of SNase is markedly shifted to highert emperatures and pressures in 30 wt %F icoll solutions,b ut the volumec hange upon unfolding is not affected significantly by crowding.…”

Elevated osmolytes in rainbow smelt: the effects of urea, glycerol and trimethylamine oxide on muscle contractile properties

“…TMAO has a negative impact on actinmyosin sliding velocity (Kumemoto et al, 2013) and LDH activity is reduced in the presence of glycerol and TMAO (Fields et al, 2001;Yancey and Siebenaller, 2015). TMAO and urea also have counteracting impacts on actin polymerization (Rosin et al, 2015), suggesting that these osmolytes may have very broad influences on the biology of smelt and other organisms that employ them for osmoregulation and cytoprotection.…”