Residue-specific multidimensional IR of cyt-c folding

描述（由申请人提供）： 三脉冲二维红外光谱（2D-IR）是一种能够解析分子结构（通过过渡偶极子和振动耦合常数之间的角度），动力学（通过可变的时间延迟和均匀的线形）和时间尺度下至飞秒的结构分布的技术。细胞色素c是一种研究充分的快速折叠血红素蛋白：在细胞色素c折叠研究中，错误折叠和配体交换结构与折叠事件相关的异质动态结构分布有关。本文提出利用二维红外光谱解析血红素附近的cyt-c肽链的平衡和动态（折叠）结构分布。Met 80（一种天然血红素配体）和Phe 82残基的13 C/180和13 C/160羰基同位素标记将用于光谱分离链的这一部分（靠近海涅）的酰胺-I振动。标记残基的2D-IR（其产生它们之间的距离和角度）将用于确定天然序列cyt-c的平衡结构分布和Phe 82突变体的扰动平衡分布。在将可见光触发脉冲集成到现有的三脉冲红外光具座中之后，变性的CO-血红素cyt-c将被光解以开始折叠成天然结构。标记残基的2D-IR将用于监测与cyt-c折叠相关的随时间演变的结构分布。

DESCRIPTION (provided by applicant): Three-pulse two-dimensional infrared spectroscopy (2D-IR) is a technique which is capable of resolving molecular structure (via angles between transition dipoles and vibrational coupling constants), dynamics (via variable time delays and homogeneous line shapes), and structural distributions on time scales down to femtoseconds. Cytochrome-c is a well-studied, fast-folding heme protein: in cyt-c folding studies, misfolded and ligand-exchanged structures have been implicated in a heterogeneous dynamic structural distribution associated with the folding events. It is proposed here to use 2D-IR to resolve the equilibrium and dynamic (folding) structural distributions of the peptide chain of cyt-c near the heme. Isotopic labeling of Met80 (a native heme ligand) and Phe82 residues with 13C/180 and 13C/160 carbonyls will be used to spectrally isolate the amide-I vibrations of this part of the chain which is close to the heine. 2D-IR of the labeled residues, which yields the distances and angles between them, will be used to determine the equilibrium structural distribution of native-sequence cyt-c and the perturbed equilibrium distributions of Phe82 mutants. After integration of a visible trigger pulse into the existing three-pulse infrared optical bench, denatured CO-heme cyt-c will be photolyzed to initiate folding to the native structure. 2D-IR of the labeled residues will be used to monitor the time-evolving structural distribution associated with cyt-c folding.