Conserved residues and their role in the structure, function, and stability of acyl-coenzyme A binding protein

In the family of acyl-coenzyme A binding proteins, a subset of 26 sequence sites are identical in all eukaryotes and conserved throughout evolution of the eukaryotic kingdoms. In the context of the bovine protein, the importa nce of these 26 sequence positions for structure, function, stability, and folding has been analyzed using single-site mutations. A total of 28 mutant proteins were analyzed which covered 17 conserved sequence positions and t hree nonconserved positions. As a first step, the influence of the mutation s on the protein folding reaction has been probed, revealing a folding nucl eus of eight hydrophobic residues formed between the N- and C-terminal heli ces [Kragelund, B. B., et al. (1999) Nat. Struct. Biol. (Ln press)]. To ful ly analyze the role of the conserved residues, the function and the stabili ty have been measured for the same set of mutant proteins. Effects on funct ion were measured by the extent of binding of the ligand dodecanoyl-CoA usi ng isothermal titration calorimetry, and effects on protein stability were measured with chemical denaturation followed by intrinsic tryptophan and ty rosine fluorescence. The sequence sites that have been conserved for direct functional purposes have been identified. These are Phe5, Tyr28, Tyr31, Ly s32, Lys54, and Tyr73. Binding site residues are mainly polar or charged re sidues, and together, four of these contribute approximately 8 kcal mol(-1) of the total free energy of binding of 11 kcal mol(-1). The sequence sites conserved for stability of the structure have likewise been identified and are Phe5, Ala9, Val12, Leu5, Leu25, Tyr28, Lys32, Gln33, Tyr73, Val77, and Leu80. Essentially, all of the conserved residues that maintain the stabil ity are hydrophobic residues at the interface of the helices. Only one cons erved polar residue, Gln33, is involved in stability. The results indicate that conservation of residues in homologous proteins may result from a summ ed optimization of an effective folding reaction, a stable native protein, and a fully active binding site. This is important in protein design strate gies, where optimization of one of these parameters, typically function or stability, may influence any of the others markedly.