Destabilised mutants of ubiquitin gain equal stability in crowded solutions

Abstract

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT AbstractThis paper investigates the thermodynamic and kinetic response of WT* ubiquitin (F45W) and three mutants to high concentrations of glucose, sucrose and dextran under physiological temperature and pH. WT* ubiquitin was stabilised by the same amount when comparing each cosolute on a weight to volume ratio, with cosolute effects largely independent of denaturant concentration. The energy difference between the mutants and WT* ubiquitin also remained the same in high concentrations of cosolute. An apparent decrease in transition-state surface burial in the presence of the cosolutes was attributed to increased compaction of the denatured state, and not to the Hammond effect. Together, these results suggest higher thermodynamic stabilities and folding rates for proteins in vivo compared to in vitro, in addition to more compact denatured states. Because the effects of mutation are the same in dilute solution and crowded conditions used to mimic the cellular environment, there is validity in using measurements of mutant stabilities made in dilute solutions to inform on how the mutations may affect stability in vivo.

Power-law dependence of the melting temperature of ubiquitin on the volume fraction of macromolecular crowders

“…This result suggests, as previously proposed, that the effect of macromolecular crowding on protein folding stability can be modeled by confinement of the protein of interest to a spherical cavity. 23 However, in contrast to theoretical prediction, but in agreement with other experimental studies, 11,14,21 we found that the stabilization arising from crowding shows a weak, if any, dependence on the size of the crowding agents used in the current study. Taken together, these findings suggest that the microscopic compartmentalization of these polymer solutions is different from what is expected based on the hydrodynamic radii of the crowders and underscore the need for further improvement of existing crowding theories.…”

“…Such a stark difference between theoretical prediction and experiment cannot be attributed to experimental uncertainty, but must arise from other reasons. Similarly, other experimental studies have also found either no 11 or a weak 14,21 dependence of the thermal stability of proteins on the crowder size. The origin of the observed discrepancy may be because when mapping a given crowded solution onto a spherical cavity via R = (4π/3ϕ) 1/3 R c , void formation due to density fluctuations in the crowded solution is not taken into account.…”

“…[1][2][3] Thus, many in vitro experiments [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] and computer simulations 10,14,15,[21][22][23][24][25][26][27][28][29] have been carried out to determine, for example, the influence of macromolecular crowding on the energy landscape of protein folding and protein-protein interactions. Additionally, several models have been developed, aiming to provide a theoretical basis for understanding the effect of macromolecular crowding on various biological events.…”

Simulation and modeling of crowding effects on the thermodynamic and kinetic properties of proteins with atomic details

“…Many experimental studies have been directed at measuring how much crowding agents affect the folding, conformational transition, and binding equilibria (Qu and Bolen 2002; Spencer et al 2005; Roberts and Jackson 2007; Batra et al 2009a; Batra et al 2009b; Phillip et al 2009; Dhar et al 2010; Miklos et al 2011; Denos et al 2012), often spurred by Minton’s predictions of significant crowder-induced stabilization of the compact states over the more open states (Minton 1981, 1998, 2000, 2005). The predictions are largely based on the scaled particle theory (Lebowitz and Rowlinson 1964) for the free energy of transferring a test protein into crowders, all of which are modeled as convex hard particles.…”

NH exchange in point mutants of human ubiquitin

“…It was shown by these authors that the conformational distortions around residue 45 are minimal and that the 3D structure is very close to the wt. Other authors [17] denoted this mutant as a pseudo wild type wt*. However, according to Roder and coworkers an enhanced flexibility exists in the region of amino acids 45-48.…”