title: Rational design, structural and thermodynamic characterization of a hyperstable variant of the villin headpiece helical subdomain.
abstract: A hyperstable variant of the small independently folded helical subdomain (HP36) derived from the F-actin binding villin headpiece was designed by targeting surface electrostatic interactions and helical propensity. A double mutant N68A, K70M was significantly more stable than wild type. The Tm of wild type in aqueous buffer is 73.0 degrees C, whereas the double mutant did not display a complete unfolding transition. The double mutant could not be completely unfolded even by 10 M urea. In 3 M urea, the Tm of wild type is 54.8 degrees C while that of the N68AK70M double mutant is 73.9 degrees C. Amide H/2H exchange studies show that the pattern of exchange is very similar for wild type and the double mutant. The structures of a K70M single mutant and the double mutant were determined by X-ray crystallography and are identical to that of the wild type. Analytical ultracentrifugation demonstrates that the proteins are monomeric. The hyperstable mutant described here is expected to be useful for folding studies of HP36 because studies of the wild type domain have sometimes been limited by its marginal stability. The results provide direct evidence that naturally occurring miniature protein domains have not been evolutionarily optimized for global stability. The stabilizing effect of this double mutant could not be predicted by sequence analysis because K70 is conserved in the larger intact headpiece for functional reasons.
authors.affiliation: Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Bi
authors.forename: Jae-Hyun
authors.initials: JH
authors.lastname: Cho
authors.forename: Eun-Young
authors.initials: EY
authors.lastname: Kim
authors.forename: Bing
authors.initials: B
authors.lastname: Shan
authors.forename: Hermann
authors.initials: H
authors.lastname: Schindelin
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Raleigh
mesh_terms: D000409:Alanine / Q000378:metabolism*; D000577:Amides / Q000737:chemistry; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011522:Protons; D012107:Research Design; D055672:Static Electricity; D013696:Temperature; D013816:Thermodynamics
chemical_list: D000577:Amides; D008840:Microfilament Proteins; D011522:Protons; C026615:villin; D000409:Alanine