The fast folding of small proteins is likely to be the product of evolutionary pressures that balance the search for native-like contacts in the transition state with the minimum number of stable non-native interactions that could lead to partially folded states prone to aggregation and amyloid formation. We have investigated the effects of non-native interactions on the folding landscape of yeast ubiquitin by introducing aromatic substitutions into the beta-turn region of the N-terminal beta-hairpin, using both the native G-bulged type I turn sequence (TXTGK) as well as an engineered 2:2 XNGK type I' turn sequence. The N-terminal beta-hairpin is a recognized folding nucleation site in ubiquitin. The folding kinetics for wt-Ub (TLTGK) and the type I' turn mutant (TNGK) reveal only a weakly populated intermediate, however, substitution with X = Phe or Trp in either context results in a high propensity to form a stable compact intermediate where the initial U-->I collapse is visible as a distinct kinetic phase. The introduction of Trp into either of the two host turn sequences results in either complex multiphase kinetics with the possibility of parallel folding pathways, or formation of a highly compact I-state stabilized by non-native interactions that must unfold before refolding. Sequence substitutions with aromatic residues within a localized beta-turn capable of forming non-native hydrophobic contacts in both the native state and partially folded states has the undesirable consequence that folding is frustrated by the formation of stable compact intermediates that evolutionary pressures at the sequence level may have largely eliminated.

Substitution of the helix-turn-helix capping motif (residues 9-35) of rabbit I-BABP with a flexible Gly-Gly-Ser-Gly linker results in the loss of stabilizing hydrophobic contacts and renders the beta-clamshell structure of this steroidal bile acid transport protein unfolded. However, in the presence of a bile acid ligand, we observe strong coupling between binding and folding, resulting in an enthalpy-driven high-affinity interaction (K(A) approximately 4 x 10(5) M(-1)) that "rescues" the native state. We investigate the mechanism of induced folding using fluorescence stopped-flow kinetic measurements to distinguish between conformational selection and induced-fit models. We observe both ligand-dependent and -independent kinetic phases which, together with their relative amplitudes, we attribute to an induced-fit "fly casting" type of model in which transient encounter complexes between the ligand and the extended polypeptide chain may act as nucleation sites for folding. An initial fast ligand-dependent kinetic process appears to be consistent with formation of a hydrophobically collapsed intermediate state which slowly rearranges to a nativelike beta-clamshell structure. We show that the intermediate forms at a rate 1000 times slower than the rate of ligand association with wild-type I-BABP, reflecting the large configurational entropic barrier to the coupled binding and folding steps of Deltaalpha-I-BABP. We have provided mechanistic insights into how natively disordered states, now commonly identified in biology, may fold on binding a target substrate or ligand.

We have investigated the relative placement of rate-limiting energy barriers and the role of productive or obstructive intermediates on the folding pathway of yeast wild-type ubiquitin ( wt-Ub) containing the F45W mutation. To manipulate the folding barriers, we have designed a family of mutants in which stabilizing substitutions have been introduced incrementally on the solvent-exposed surface of the main alpha-helix (residues 23-34), which has a low intrinsic helical propensity in the native sequence. Although the U --> I and I --> N transitions are not clearly delineated in the kinetics of wt-Ub, we show that an intermediate becomes highly populated and more clearly resolved as the predicted stability of the helix increases. The observed acceleration in the rate of folding correlates with helix stability and is consistent with the I-state representing a productive rather than misfolded state. A Leffler analysis of the effects on kinetics of changes in stability within the family of helix mutants results in a biphasic correlation in both the refolding and unfolding rates that suggest a shift from a nucleation-condensation mechanism (weakly stabilized helix) toward a diffusion-collision model (highly stabilized helix). Through the introduction of helix-stabilizing mutations, we are able to engineer a well-resolved I-state on the folding pathway of ubiquitin which is likely to be structurally distinct from that which is only weakly populated on the folding pathway of wild-type ubiquitin.

New mechanistic understanding and the quantification of reaction kinetics shed light on the large impact of the solvent on selectivity.