Side-chain and backbone flexibility in protein core design

We have developed a computational approach for the design and prediction of hydrophobic cores that includes explicit backbone flexibility. The program consists of a two-stage combination of a genetic algorithm and monte carlo sampling using a torsional model of the protein. Backbone structures are e valuated either by a canonical force-field or a constraining potential that emphasizes the preservation of local geometry. The utility of the method f or protein design and engineering is explored by designing three novel hydr ophobic core variants of the protein 434 cro. We use the new method to eval uate these and previously designed 434 cro variants, as well as a series of phage T4 lysozyme variants. In order to properly evaluate the influence of backbone flexibility, we have also analyzed the effects of varying amounts of side-chain flexibility on the performance of fixed back,bone methods. C omparison of results using a fixed versus flexible backbone reveals that, s urprisingly, the two methods are almost equivalent in their abilities to pr edict relative experimental stabilities, but only when full side-chain flex ibility is allowed. The prediction of core side-chain structure can vary dr amatically between methods. Ln some, but not all, cases the flexible backbo ne method is a better predictor of structure. The development of a flexible backbone approach to core design is particularly important for attempts at de novo protein design, where there is no prior knowledge of a precise bac kbone structure. (C) 1999 Academic Press.