Dramatic stabilization of an SH3 domain by a single substitution: roles of the folded and unfolded states.
The N-terminal SH3 domain of the Drosophila drk protein (drkN SH3) exists in equilibrium between folded and unfolded states under non-denaturing buffer conditions. In order to examine the origins of this instability, we have made mutations in the domain and characterized the thermodynamics and kinetics of folding. Results of substitutions of negatively charged residues to neutral amino acid residues suggest that the large electrostatic potential of the domain does not play a dominant role in the instability of the domain. Sequence alignment of a large number of SH3 domains reveals that the drkN SH3 domain has a threonine (T22) at a position corresponding to an otherwise highly conserved glycine residue in the diverging beta-turn connecting the beta3 and beta4 strands. Mutation of T22 to glycine results in significant stabilization of the drkN SH3 domain by 2.5 kcal/mole. To further characterize the basis for the stabilization of the T22 mutant relative to wild-type, we made additional mutant proteins with substitutions of residue T22. A strong correlation is seen between protein stability or folding rate and propensity for native beta-turn structure at this position. Correlation of folding rates with AGADIR predictions of non-native helical structure in the diverging turn region, along with our previous NMR evidence for non-native structure in this region of the unfolded state of the drkN SH3 domain, suggests that the free energy of the unfolded state also plays a role in stability. This result highlights the importance of both folded and unfolded states for understanding protein stability.
Mok
Y K
YK
Department of Biochemistry, Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada.
Elisseeva
E L
EL
Davidson
A R
AR
Forman-Kay
J D
JD
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002942:Circular Dichroism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000737:chemistry* / Q000235:genetics; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects; D055672:Static Electricity; D013696:Temperature; D013816:Thermodynamics; D014466:Ultraviolet Rays; D018909:src Homology Domains / Q000187:drug effects
D029721:Drosophila Proteins; D019476:Insect Proteins; C080530:drk protein, Drosophila; D019791:Guanidine