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

Abstract

Actin polymerization is an essential process in eukaryotic cells that provides a driving force for motility and mechanical resistance for cell shape. By using preformed gelsolin-actin nuclei and applying stopped-flow methodology, we quantitatively studied the elongation kinetics of actin filaments as a function of temperature and pressure in the presence of synthetic and protein crowding agents. We show that the association of actin monomers to the pointed end of double-stranded helical actin filaments (F-actin) proceeds via a transition state that requires an activation energy of 56 kJ mol(-1) for conformational and hydration rearrangements, but exhibits a negligible activation volume, pointing to a compact transition state that is devoid of packing defects. Macromolecular crowding causes acceleration of the F-actin elongation rate and counteracts the deteriorating effect of pressure. The results shed new light on the combined effect of these parameters on the polymerization process of actin, and help us understand the temperature and pressure sensitivity of actin polymerization under extreme conditions.

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.…”

“…In the reaction‐controlled regime, crowding is expected to effectively lower the energy barrier and thus increase the association rate constant, because the transition state for association in solution is generally nearly as compact as the product complex . Solutions crowded with synthetic polymers and the protein BSA have been reported to retard the growth of amyloid fibrils due to pronounced binding behavior to the monomeric protein, whereas the elongation of cytoskeletal actin filaments has been shown to be accelerated under the same crowding conditions …”

“…[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.…”

“…[8] Solutions crowded with synthetic polymers and the protein BSA have been reported to retard the growth of amyloid fibrils due to pronouncedb inding behavior to the monomeric protein, [219] whereas the elongation of cytoskeletal actin filaments has been shown to be accelerated under the same crowding conditions. [201] In the case of protein folding, unfolded conformations are more expanded and thus crowding is expected to favor the . ChemPhysChem 2017ChemPhysChem , 18,2951ChemPhysChem -2972 www.chemphyschem.org native state and hence the folding rate.…”

Modulation of the Polymerization Kinetics of α/β‐Tubulin by Osmolytes and Macromolecular Crowding

“…Additional enthalpic effects of a weak nature might also be involved to cause the decrease in the rate of the polymerization process . A similar effect of sucrose was found for the elongation reaction of actin filaments and the fibrillation reaction of human islet amyloid polypeptide (hIAPP) . The comparative use of Ficoll 70 and sucrose reveals that the accelerative effect of macromolecular crowding is predominantly caused by the excluded volume effect of Ficoll 70 and, thus, the elevated effective protein concentration rather than by changes in viscosity or the effects of enthalpic nature (weak protein–crowder interactions) …”