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Abstract

Making use of an ab-initio folding simulator, we generate in vitro pathways leading to the native fold in moderate size single-domain proteins. The assessment of pathway diversity is not biased by any a-priori information on the native fold. We focus on two study cases, hyperthermophile variant of protein G domain (1gb4) and ubiquitin (lubi), with the same topology but different context dependence in their native folds. We demonstrate that a quenching of structural fluctuations is achieved once the proteins find a stationary plateau maximizing the number of highly protected hydrogen bonds. This enables us to identify the folding nucleus and show that folding does not become expeditious until a concerted event takes place generating a topology able to prevent water attack on a maximal number of hydrogen bonds. This result is consistent with the standard nucleation mechanism postulated for two-state folders. Pathway diversity is correlated with the extent of conflict between local structural propensity and large-scale context, rather than with contact order: In highly context-dependent proteins, the success of folding cannot rely on a single fortuitous event in which local propensity is overruled by large-scale effects. We predict mutational (D values on individual pathways, compute ensemble averages and predict extent of surface burial and percentage of hydrogen bonding on each component of the transition state ensemble, thus deconvoluting individual folding-route contributions to the averaged two-state kinetic picture. Our predicted kinetic isotopic effects find experimental support and lead to further probes. Finally, the molecular redesign potentiality of the method, aimed at increasing folding expediency, is explored.