Effects of pH, salt, and macromolecular crowding on the stability of FK506-binding protein: an integrated experimental and theoretical study.
Environmental variables can exert significant influences on the folding stability of a protein, and elucidating these influences provides insight on the determinants of protein stability. Here, experimental data on the stability of FKBP12 are reported for the effects of three environmental variables: pH, salt, and macromolecular crowding. In the pH range of 5-9, contribution to the pH dependence of the unfolding free energy from residual charge-charge interactions in the unfolded state was found to be negligible. The negligible contribution was attributed to the lack of sequentially nearest neighboring charged residues around groups that titrate in the pH range. KCl lowered the stability of FKBP12 and the E31Q/D32N double mutant at small salt concentrations but raised stability after approximately 0.5 M salt. Such a turnover behavior was accounted for by the balance of two opposing types of protein-salt interactions: the Debye-Hückel type, modeling the response of the ions to protein charges, favors the unfolded state while the Kirkwood type, accounting for the disadvantage of the ions moving toward the low-dielectric protein cavity from the bulk solvent, disfavors the unfolded state. Ficoll 70 as a crowding agent was found to have a modest effect on protein stability, in qualitative agreement with a simple model suggesting that the folded and unfolded states are nearly equally adversely affected by macromolecular crowding. For any environmental variable, it is the balance of its effects on the folded and unfolded states that determines the outcome on the folding stability.
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA.
Daniel S
DS
Spencer
Ke
K
Xu
Timothy M
TM
Logan
Huan-Xiang
HX
Zhou
D005362:Ficoll / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011108:Polymers / Q000494:pharmacology*; D011189:Potassium Chloride / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding; D012492:Salts / Q000494:pharmacology*; D055672:Static Electricity; D022021:Tacrolimus Binding Proteins / Q000737:chemistry*
D011108:Polymers; D012492:Salts; D005362:Ficoll; D011189:Potassium Chloride; D022021:Tacrolimus Binding Proteins