SYNTHETIC AND BIOLOGICAL STUDIES OF NUCLEIC ACIDS

I.细菌视紫红质：细菌视紫红质，是 嗜盐杆菌分化的紫膜，光传导 通过从内部向外部泵送质子将能量转化为化学能 手机。这项工作的目的是了解质子的机制。 这种膜蛋白的易位。该机制是否涉及 质子通道或沿质子“线”的导电性？实验性的 这种方法将涉及蛋白质中特定的氨基酸替换。 才能提出具体的问题。突变蛋白将按位置制备 细菌视紫红质基因的特异性突变及表达。 将检查它们的以下性质：(1)重折叠、结合 视网膜和细菌视紫红质样发色团的再生； 重组为囊泡和泵入质子的能力；(3)生物物理 傅里叶变换红外激光拉曼光谱研究 突变对蛋白质结构、蛋白质间相互作用的影响 视网膜和蛋白质在光化学循环的不同步骤上； 以及(4)光化学循环效应与光化学循环效应之间的可能相关性。 质子转移。 II.脊椎动物杆状外节中光传导的生物化学 视网膜；视紫红质的结构-功能研究。一个主要目标是 了解视紫红质的动态变化；漂白的结构变化， 与GTP酶的相互作用及磷酸化和蛋白水解酶的调节 去磷酸化。视紫红质在膜水平的研究将涉及 脱脂、变性、折叠和重组。一个主要目标 将是通过特定部位来研究结构-功能关系 基因的诱变和突变基因产物的表达。总计 视紫红质基因的合成已经进行，以便于 在整个基因中进行完全不受限制的突变。为此目的， 适当数目的独特限制地点已在 基因上的适当位置。棒材外段的结构 参与光信号转导的蛋白质(GTP酶、cGMP磷酸二酯酶、 Na通道)也在用重组DNA的方法进行研究。

I. BACTERIORHODOPSIN: Bacteriorhodopsin, the single protein of the differentiated purple membrane in Halobacterium halobium, transduces light energy to chemical energy by pumping protons from inside to outside of the cell. The purpose of this work is to understand the mechanism of proton translocation by this membrane protein. Does the mechanism involve a proton channel or conductance along a proton "wire"? The experimental approach would involve specific amino acid replacements in the protein in order to ask specific questions. Mutant proteins will be prepared by site specific mutagenesis of the bacteriorhodopsin gene followed by expression. They will be examined for the following properties: (1) refolding, binding of retinal and regeneration of bacteriorhodopsin-like chromophore; (2) reconstitution into vesicles and ability to pump protons; (3) biophysical (Fourier transform infrared laser Raman spectroscopy) studies of the effects of mutations on the protein structure, on interactions between retinal and the protein and on different steps in the photochemical cycle; and (4) possible correlations between effects on photochemical cycle and on proton translocation. II. BIOCHEMISTRY OF LIGHT-TRANSDUCTION IN ROD OUTER SEGMENTS IN VERTEBRATE RETINA; STRUCTURE-FUNCTION STUDIES ON RHODOPSIN. A major objective is to understand the dynamics of rhodopsin; the structural change on bleaching, the interaction with GTPase and the regulation of phosphorylation and dephosphorylation. Studies of rhodopsin at membrane level would involve delipidation, denaturation, refolding and reconstitution. A major aim would be the study of structure-function relationships by site-specific mutagenesis of the gene and expression of the mutated gene products. Total synthesis of the rhodopsin gene has been undertaken so as to facilitate completely unrestricted mutagenesis throughout the gene. For this purpose a suitable number of unique restriction sites have been introduced at appropriate positions along the gene. Structures of the rod outer segment proteins involved in light transduction (GTPase, cGMP phosphodiesterase, the Na channel) are also being investigated by methods of recombinant DNA.