Cosolvent and Crowding Effects on the Temperature‐ and Pressure‐Dependent Dissociation Process of the α/β‐Tubulin Heterodimer

Abstract

Tubulin is one of the main components of the cytoskeleton of eukaryotic cells. The formation of microtubules depends strongly on environmental and solution conditions, and has been found to be among the most pressure sensitive processes in vivo. We explored the effects of different types of cosolvents, such as trimethylamine‐N‐oxide (TMAO), sucrose and urea, and crowding agents to mimic cell‐like conditions, on the temperature and pressure stability of the building block of microtubules, i. e. the α/β‐tubulin heterodimer. To this end, fluorescence and FTIR spectroscopy, differential scanning and pressure perturbation calorimetry as well as fluorescence anisotropy and correlation spectroscopies were applied. The pressure and temperature of dissociation of α/β‐tubulin as well as the underlying thermodynamic parameters upon dissociation, such as volume and enthalpy changes, have been determined for the different solution conditions. The temperature and pressure of dissociation of the α/β‐tubulin heterodimer and hence its stability increases dramatically in the presence of TMAO and the nanocrowder sucrose. We show that by adjusting the levels of compatible cosolutes and crowders, cells are able to withstand deteriorating effects of pressure even up to the kbar‐range.

Biomolecules under Pressure: Phase Diagrams, Volume Changes, and High Pressure Spectroscopic Techniques

“…Actin is one of the most basic proteins in all living organisms. Globular actin is stable up to 270 MPa at room temperature [ 112 ], but polymerization of the actin monomers is known to be a very pressure-sensitive process; even pressures of a few hundred bar lead to a significant deceleration of the polymerization reaction, mainly due to the prolonged nucleation phase [ 113 ]. Morita found amino acid substitutions which can also contribute to the deep-sea adaptation of actin [ 114 ].…”

Deep sea osmolytes in action: their effect on protein–ligand binding under high pressure stress

“…27,28 It has also been shown to enhance the assembly of the eukaryotic cytoskeletal protein tubulin. 29 Glycine betaine (GB) is also a naturally-occurring osmolyte, responsible for maintaining osmotic balance of environmentally stressed cells. 22–24 Additionally, like other osmoprotectants, GB can act as a protectant, stabilizing the native structure of proteins and counteracting the denaturing effect of destabilizing cosolutes.…”

Pressure Tuning Studies of Four-Stranded Nucleic Acid Structures

“…This effect is called macromolecular crowding [ 22 ]. This has a pronounced effect on the stability of proteins, nucleic acids and on cellular processes [ 21 , 23 , 24 , 25 , 26 ].…”