title: The osmolyte trimethylamine-N-oxide stabilizes the Fyn SH3 domain without altering the structure of its folding transition state.
abstract: Trimethylamine-N-oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins against denaturation. Although the impact of TMAO on the folding thermodynamics of many proteins has been well characterized, far fewer studies have investigated its effects on protein folding kinetics. In particular, no previous studies have used Phi-value analysis to determine whether TMAO may alter the structure of the folding transition state. Here we have measured the effects on folding kinetics of 16 different amino acid substitutions distributed across the structure of the Fyn SH3 domain both in the presence and absence of TMAO. The folding and unfolding rates in TMAO, on average, improved to equivalent degrees, with a twofold increase in the protein folding rate accompanied by a twofold decrease in the unfolding rate. Importantly, TMAO caused little alteration to the Phi-values of the mutants tested, implying that this compound minimally perturbs the folding transition state structure. Furthermore, the solvent accessibility of the transition state was not altered as reflected in an absence of a TMAO-induced change in the denaturant beta(T) (D) factors. Through TMAO-induced folding studies, a beta(T) (TMAO) factor of 0.5 was calculated for this compound, suggesting that the protein backbone, which is the target of action of TMAO, is 50% exposed in the transition state as compared to the native state. This finding is consistent with the equivalent effects of TMAO on the folding and unfolding rates. Through thermodynamic analysis of mutants, we also discovered that the stabilizing effect of TMAO is lessened with increasing temperature.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Sung Lun
authors.initials: SL
authors.lastname: Lin
authors.forename: Arash
authors.initials: A
authors.lastname: Zarrine-Afsar
authors.forename: Alan R
authors.initials: AR
authors.lastname: Davidson
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics / Q000502:physiology; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D008744:Methylamines / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D018909:src Homology Domains / Q000235:genetics* / Q000502:physiology
chemical_list: D008744:Methylamines; D051076:Proto-Oncogene Proteins c-fyn; C005855:trimethyloxamine; D019791:Guanidine