FAST AND ONE-STEP FOLDING OF CLOSELY AND DISTANTLY RELATED HOMOLOGOUSPROTEINS OF A 4-HELIX BUNDLE FAMILY

Bovine acyl-coenzyme A binding protein is a four-helix bundle protein belonging to a group of homologous eukaryote proteins that binds mediu m and long-chain acyl-coenzyme A esters with a very high affinity. The three-dimensional structure of both the free and the ligated protein together with the folding kinetics have been described in detail for t he bovine protein and with four new sequences reported here, a total o f 16 closely related sequences ranging from yeasts and plants to human are known. The kinetics of folding and unfolding in different concent rations of guanidine hydrochloride together with equilibrium unfolding have been measured for bovine, rat and yeast acyl-coenzyme A binding protein. The bovine and rat sequences are closely related whereas the yeast is more distantly related to these. In addition to the three nat ural variants, kinetics of a bovine mutant protein, Tyr31 --> Asn, hav e been studied. Both the folding and unfolding rates in water of the y east protein are 15 times faster than those of bovine. The folding rat es in water of the two mammalian forms, rat and bovine, are similar, t hough still significantly different. A faster unfolding rate both for rat and the bovine mutant protein results from a lower stability of th e native states of these. These hydrophobic regions, mini cores, have been identified in the three-dimensional structure of the bovine prote in and found to be formed primarily by residues that have been conserv ed throughout the entire eukaryote evolution from yeasts to both plant s and mammals as seen in the sample of 16 sequences. The conserved res idues are found to stabilize helix-helix interactions and serve specif ic functional purposes for ligand binding. The fast one-step folding m echanism of ACBP has been shown to be a feature that seems to be maint ained throughout evolution despite numerous differences in sequence an d even dramatic differences in folding kinetics and protein stability. The protein study raises the question to what extent does the conserv ed hydrophobic residues provide a scaffold for an efficient one-step f olding mechanism. (C) 1996 Academic Press Limited.