Barua Bipasha, Lin Jasper C, Williams Victoria D, Kummler Phillip, Neidigh Jonathan W, Andersen Niels H
Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
Protein Eng Des Sel. 2008 Mar;21(3):171-85. doi: 10.1093/protein/gzm082. Epub 2008 Jan 18.
The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (+/-0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (DeltaDeltaS(U) = 11 +/- 2 J/mol K) was measured at the solvent-exposed P17 site. Pro-Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3-3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4-6 kJ/mol) and a buried H-bonded Ser side chain ( approximately 10 kJ/mol).
作为最小的微型蛋白质，色氨酸笼仍然是蛋白质折叠动力学和途径的众多计算和实验研究的对象。原始的色氨酸笼（NLYIQWLKDGGPSSGRPPPS，熔点Tm = 42摄氏度）可以通过突变得到显著稳定；据报道熔点高达64摄氏度。在该结构的螺旋部分，亮氨酸、异亮氨酸、赖氨酸或丝氨酸残基每次被丙氨酸取代都会导致折叠稳定性增加1.5（±0.35）kJ/mol。稳定后核心结构或流动性没有变化。与最初的假设相反，特定的脯氨酸/色氨酸相互作用对于核心形成并非必不可少。在溶剂暴露的P17位点测量了脯氨酸相对于丙氨酸的熵优势（ΔΔS(U)= 11±2 J/mol K）。包围吲哚环的三个脯氨酸中的两个（P12和P18）发生脯氨酸-丙氨酸突变导致折叠稳定性降低较少（2.3 - 3.4 kJ/mol）。然而，P19A突变使折叠稳定性降低16 kJ/mol，这反映了Y3/P19的有利相互作用以及色氨酸的埋藏。Y3/P19疏水主链相互作用将折叠基序定义为一个18个残基的单元。已确定的其他稳定特征包括一个溶剂暴露的精氨酸/天冬氨酸盐桥（3.4 - 6 kJ/mol）和一个埋藏的氢键连接的丝氨酸侧链（约10 kJ/mol）。
