Ionic-strength-dependent effects in protein folding: analysis of rate equilibrium free-energy relationships and their interpretation.
The traditional approach to studying protein folding involves applying a perturbation, usually denaturant or mutation, and determining the effect upon the free energy of folding, DeltaG0, and the activation free energy, DeltaG(not equal). Data collected as a function of the perturbation can be used to construct rate equilibrium free-energy relationships, which report on the development of interactions in the transition state for folding. We examine the use of the ionic-strength-dependent rate equilibrium free-energy relationship in protein folding using the N-terminal domain of L9, a small alpha-beta protein, as a model system. Folding is two-state for the range of ionic strength examined, 0.045-1.52 M. The plot of DeltaG(not equal) versus DeltaG0 is linear (r2= 0.918), with a slope equal to 0.45. The relatively low value of the slope indicates that the ionic-strength-dependent interactions are modestly developed in the transition state. The slope is, however, greater than that of a plot of DeltaG(not equal) versus DeltaG0 constructed by varying pH, thus demonstrating directly that ionic-strength-dependent studies probe more than simple electrostatic interactions. Potential transition movement was probed by analysis of the denaturant, ionic strength cross-interaction parameters. The values are small but nonzero and positive, suggesting a small shift of the transition state toward the native state as the protein is destabilized, i.e., Hammond behavior. The complications that arise in the interpretation of ionic-strength-dependent rate equilibrium free-energy relationships are discussed, and it is concluded that the ionic-strength-dependent studies do not provide a reliable indicator of the role of electrostatic interactions. Complications include incomplete screening of electrostatic interactions, specific ion binding, Hofmeister effects, and the potential presence of electrostatic interactions in the denatured state ensemble.
Song
Benben
B
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, USA.
Cho
Jae-Hyun
JH
Raleigh
Daniel P
DP
D007700:Kinetics; D008958:Models, Molecular; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D012269:Ribosomal Proteins / Q000737:chemistry*; D013816:Thermodynamics
D010446:Peptide Fragments; D012269:Ribosomal Proteins; C049650:ribosomal protein L9