Native state energetics of the Src SH2 domain: evidence for a partially structured state in the denatured ensemble.
We have defined the free-energy profile of the Src SH2 domain using a variety of biophysical techniques. Equilibrium and kinetic experiments monitored by tryptophan fluorescence show that Src SH2 is quite stable and folds rapidly by a two-state mechanism, without populating any intermediates. Native state hydrogen-deuterium exchange confirms this two-state behavior; we detect no cooperative partially unfolded forms in equilibrium with the native conformation under any conditions. Interestingly, the apparent stability of the protein from hydrogen exchange is 2 kcal/mol greater than the stability determined by both equilibrium and kinetic studies followed by fluorescence. Native-state proteolysis demonstrates that this "super protection" does not result from a deviation from the linear extrapolation model used to fit the fluorescence data. Instead, it likely arises from a notable compaction in the unfolded state under native conditions, resulting in an ensemble of conformations with substantial solvent exposure of side chains and flexible regions sensitive to proteolysis, but backbone amides that exchange with solvent approximately 30-fold slower than would be expected for a random coil. The apparently simple behavior of Src SH2 in traditional unfolding studies masks the significant complexity present in the denatured-state ensemble.
Wildes
David
D
Department of Molecular and Cell Biology, University of California, Berkeley 94720-3206, USA.
Anderson
L Meadow
LM
Sabogal
Alex
A
Marqusee
Susan
S
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002645:Chickens; D041961:Deuterium Exchange Measurement; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan; D014508:Urea / Q000494:pharmacology; D018909:src Homology Domains; D019061:src-Family Kinases
D014364:Tryptophan; D014508:Urea; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases