The ability to finely control the structure of protein folds is an important prerequisite to functional protein design. The TIM barrelfold is an important target for these efforts as it is highly enriched for diverse ...The ability to finely control the structure of protein folds is an important prerequisite to functional protein design. The TIM barrelfold is an important target for these efforts as it is highly enriched for diverse functions in nature. Although a TIM barrel proteinhas been designed de novo, the ability to finely alter the curvature of the central beta barrel and the overall architecture of the foldremains elusive, limiting its utility for functional design. Here, we report the de novo design of a TIM barrel with ovoid (twofold)symmetry, drawing inspiration from natural beta and TIM barrels with ovoid curvature. We use an autoregressive backbonesampling strategy to implement our hypothesis for elongated barrel curvature, followed by an iterative enrichment sequencedesign protocol to obtain sequences which yield a high proportion of successfully folding designs. Designed sequences arehighly stable and fold to the designed barrel curvature as determined by a 2.1Å resolution crystal structure. The designs showrobustness to drastic mutations, retaining high melting temperatures even when multiple charged residues are buried in thehydrophobic core or when the hydrophobic core is ablated to alanine. As a scaffold with a greater capacity for hosting diversehydrogen bonding networks and installation of binding pockets or active sites, the ovoid TIM barrel represents a major steptowards the de novo design of functional TIM barrels.展开更多
基金the U.S.Department of Energy,Office of Science,and Office of Basic Energy Sciences under Contract No.DE-AC02-76SF00515The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health,National Institute of General Medical Sciences(including P41GM103393).A.E.C.is supported by the National Science Foundation Graduate Research Fellowship.
文摘The ability to finely control the structure of protein folds is an important prerequisite to functional protein design. The TIM barrelfold is an important target for these efforts as it is highly enriched for diverse functions in nature. Although a TIM barrel proteinhas been designed de novo, the ability to finely alter the curvature of the central beta barrel and the overall architecture of the foldremains elusive, limiting its utility for functional design. Here, we report the de novo design of a TIM barrel with ovoid (twofold)symmetry, drawing inspiration from natural beta and TIM barrels with ovoid curvature. We use an autoregressive backbonesampling strategy to implement our hypothesis for elongated barrel curvature, followed by an iterative enrichment sequencedesign protocol to obtain sequences which yield a high proportion of successfully folding designs. Designed sequences arehighly stable and fold to the designed barrel curvature as determined by a 2.1Å resolution crystal structure. The designs showrobustness to drastic mutations, retaining high melting temperatures even when multiple charged residues are buried in thehydrophobic core or when the hydrophobic core is ablated to alanine. As a scaffold with a greater capacity for hosting diversehydrogen bonding networks and installation of binding pockets or active sites, the ovoid TIM barrel represents a major steptowards the de novo design of functional TIM barrels.
