Crystal structures and increased stabilization of the protein G variants with switched folding pathways NuG1 and NuG2.
We recently described two protein G variants (NuG1 and NuG2) with redesigned first hairpins that were almost twice as stable, folded 100-fold faster, and had a switched folding mechanism relative to the wild-type protein. To test the structural accuracy of our design algorithm and to provide insights to the dramatic changes in the kinetics and thermodynamics of folding, we have now determined the crystal structures of NuG1 and NuG2 to 1.8 A and 1.85 A, respectively. We find that they adopt hairpin structures that are closer to the computational models than to wild-type protein G; the RMSD of the NuG1 hairpin to the design model and the wild-type structure are 1.7 A and 5.1 A, respectively. The crystallographic B factor in the redesigned first hairpin of NuG1 is systematically higher than the second hairpin, suggesting that the redesigned region is somewhat less rigid. A second round of structure-based design yielded new variants of NuG1 and NuG2, which are further stabilized by 0.5 kcal/mole and 0.9 kcal/mole.
Nauli
Sehat
S
Department of Biochemistry, University of Washington, Seattle 98195, USA.
Kuhlman
Brian
B
Le Trong
Isolde
I
Stenkamp
Ronald E
RE
Teller
David
D
Baker
David
D
D018360:Crystallography, X-Ray; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D013816:Thermodynamics
C029152:G-substrate; D009419:Nerve Tissue Proteins