Ultraviolet Resonance Raman Spectroscopic Study on Regulatory Protein-Nucleic Acid Interactions

本研究的目的有三个方面：紫外拉曼光谱仪的建立，蛋白质和核酸结构研究中有用的紫外拉曼标记谱带的列表，以及溶液中环腺苷酸受体蛋白（CRP）及其复合物的紫外共振拉曼光谱研究。(1)紫外拉曼装置紫外辐射由Nd：YAG激光器（30 Hz）、谐波发生器（KDP或BBO）和H_2-拉曼位移器组成。来自样品的散射光通过f/6.8 80-cm双单色仪分散（第二阶），该双单色仪配备有在500 nm处闪耀的600 g/mm光栅，并用700像素的双增强二极管阵列检测。(2)UVRR标记带表及其应用含有Pro、Trp、Tyr、Phe和His的肽、核苷、核苷酸和相关化合物的UVRR光谱已被广泛研究，并列出了有用的结构标记带列表。首次记录了Cu，Zu-超氧化物歧化酶和细菌视紫红质的高质量UVRR光谱，并根据该表获得了一些蛋白质的结构信息。(3)CRP和CRP-cAMP在稀溶液中的结构当CRP的两个亚基之一摄取cAMP分子时，另一个亚基以及结合的亚基的β折叠结构发生剧烈变化。结合cAMP的腺嘌呤-核糖键构象为顺式，结合形式通过N7和C6NH_2与蛋白质侧链的氢键而稳定。

The purpose of the research is threefold : construction of ultraviolet Raman apparatus, tabulation of UV Raman marker bands useful in structural study of proteins and nucleic acids, and UV resonance Raman (UVRR) spectroscopic study on cyclic AMP receptor protein (CRP) and the CRP-cAMP complex in solution.(1) UV Raman apparatusUV radiation is generated by a system consisting of a Nd : YAG laser (30 Hz), a harmonic generator (KDP or BBO), and an H_2-Raman shifter. Scattered light from a sample is dispersed (2nd order) by an f/6.8 80-cm double monochromator equipped with 600 g/mm gratings blazed at 500 nm and detected with a doubly intensified diode array of 700 pixels.(2) Table of UVRR marker bands and its applicationsUVRR spectra of peptides containing Pro, Trp, Tyr, Phe, and His, nucleosides, nucleotides, and the related compounds have been studied extensively and a list of useful structural marker bands is tabulated. Good-quality UVRR spectra of Cu, Zu-superoxide dismutase and bacteriorhodopsin have been recorded for the first time, and some pieces of structural information on the proteins are obtained on the basis of the table.(3) Structure of CRP and CRP-cAMP in dilute solutionUpon uptake of a cAMP molecule in one of the two subunits of CRP, drastic change occurs in the beta-sheet structure of the other subunit as well as the bound subunit. The adenine-ribose bond conformation is syn in the bound cAMP and the binding form is stabilized by H-bonding at N7 and C6NH_2 with side chains of the protein.