Conserved Residues and Their Role in the Structure, Function, and Stability of Acyl-Coenzyme A Binding Protein

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• 作者：Birthe B. Kragelund ; Keld Poulsen ; Kim Vilbour Andersen ; Trausti Baldursson ; Jenny B. Kr//pubs.acs.org/images/gifchars/oslash.gif" border="0">ll ; Thomas B. Neergå ; rd ; Jan Jepsen ; P
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：February 23, 1999
• 年：1999
• 卷：38
• 期：8
• 页码：2386 - 2394
• 全文大小：142K
• 年卷期：v.38,no.8(February 23, 1999)
• ISSN：1520-4995

文摘

In the family of acyl-coenzyme A binding proteins, a subset of 26 sequence sites are identicalin all eukaryotes and conserved throughout evolution of the eukaryotic kingdoms. In the context of thebovine protein, the importance of these 26 sequence positions for structure, function, stability, and foldinghas been analyzed using single-site mutations. A total of 28 mutant proteins were analyzed which covered17 conserved sequence positions and three nonconserved positions. As a first step, the influence of themutations on the protein folding reaction has been probed, revealing a folding nucleus of eight hydrophobicresidues formed between the N- and C-terminal helices [Kragelund, B. B., et al. (1999) Nat. Struct. Biol.(In press)]. To fully analyze the role of the conserved residues, the function and the stability have beenmeasured for the same set of mutant proteins. Effects on function were measured by the extent of bindingof the ligand dodecanoyl-CoA using isothermal titration calorimetry, and effects on protein stability weremeasured with chemical denaturation followed by intrinsic tryptophan and tyrosine fluorescence. Thesequence sites that have been conserved for direct functional purposes have been identified. These arePhe5, Tyr28, Tyr31, Lys32, Lys54, and Tyr73. Binding site residues are mainly polar or charged residues,and together, four of these contribute approximately 8 kcal mol^-1 of the total free energy of binding of11 kcal mol^-1. The sequence sites conserved for stability of the structure have likewise been identifiedand are Phe5, Ala9, Val12, Leu15, Leu25, Tyr28, Lys32, Gln33, Tyr73, Val77, and Leu80. Essentially,all of the conserved residues that maintain the stability are hydrophobic residues at the interface of thehelices. Only one conserved polar residue, Gln33, is involved in stability. The results indicate thatconservation of residues in homologous proteins may result from a summed optimization of an effectivefolding reaction, a stable native protein, and a fully active binding site. This is important in protein designstrategies, where optimization of one of these parameters, typically function or stability, may influenceany of the others markedly.