Rescuing a destabilized protein fold through backbone cyclization.
We describe the physicochemical characterization of various circular and linear forms of the approximately 60 residue N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein. Structural, dynamic, thermodynamic, kinetic and biochemical studies reveal that backbone circularization does not prevent the adoption of the natural folded structure in any of the circular proteins. Both the folding and unfolding rate of the protein increased slightly upon circularization. Circularization did not lead to a significant thermodynamic stabilization of the full-length protein, suggesting that destabilizing enthalpic effects (e.g. strain) negate the expected favorable entropic contribution to overall stability. In contrast, we find circularization results in a dramatic stabilization of a truncated version of the SH3 domain lacking a key glutamate residue. The ability to rescue the destabilized mutant indicates that circularization may be a useful tool in protein engineering programs geared towards generating minimized proteins.
Camarero
J A
JA
The Laboratory of Synthetic Protein Chemistry, New York, NY 10021, USA.
Fushman
D
D
Sato
S
S
Giriat
I
I
Cowburn
D
D
Raleigh
D P
DP
Muir
T W
TW
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003500:Cyclization; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation / Q000187:drug effects; D015202:Protein Engineering; D017510:Protein Folding; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics; D050737:Proto-Oncogene Proteins c-crk; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology; D018909:src Homology Domains / Q000187:drug effects
D008024:Ligands; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D018698:Glutamic Acid; D014508:Urea