Folding of a predominantly beta sheet protein with a central cavity

The structure of cellular retinoic acid binding protein I ((!RABPI) is composed of a short helix-turn-helix and two nearly orthogona] five-strand beta sheets which surround a central cavity. Kinetic analysis by stopped-flow fluorescence of native and sing]e-Trp variants, hydrogen exchange, and probing of ligand binding provides a description of the pathway of refolding of CRABPI. An early phase ( I0 ms) leads to a hydrophobically collapsed state with nativelike helical structure. Native-like topology', as indicated by ]]sand-binding capacity and Trp fluorescence, develops in a ca. I00 ms phase. Strikingly, stable hydrogen bonding forms in the beta sheets in a fully cooperative manner in a later (I s) phase, during which specific packing interactions (as indicated by" fluorescence) also develop. These resnlts have implications for the formation of beta sheets, which is poorly understood at present, and the determination of topology, viz. when and how does the central cavity form. We conclude thnt the topoloO of the beta sheets in CRABPI is guided by incipient native side chain interactions; stable hydrogen bonding and presumably, solvent exclusion, occur only during the final stage in folding.