SYNTHETIC AND BIOLOGICAL STUDY OF NUCLEIC ACIDS

I.细菌视紫红质：细菌视紫红质， 在盐生盐杆菌中分化的紫色膜， 通过从内部到外部泵送质子， cell. 本工作的目的是了解质子的机制 通过这种膜蛋白转运。 该机制是否涉及 质子通道或质子传导沿着“线”？ 实验 这种方法将涉及蛋白质中特定的氨基酸替换， 为了提出具体的问题。 突变蛋白将按研究中心制备 细菌视紫红质基因的特异性诱变，随后表达。 它们将被检查以下性质：（1）重折叠，结合 （2）视网膜再生与细菌视紫红质样发色团再生; 重组成囊泡和泵送质子的能力;（3）生物物理 （傅里叶变换红外激光拉曼光谱）的研究 突变对蛋白质结构的影响， 视黄酸和蛋白质以及光化学循环中的不同步骤; （4）对光化学循环和对生物体的影响之间可能存在的相关性。 质子移位 二.脊椎动物视杆细胞外节光传导的生物化学 视网膜;视紫红质的结构-功能研究。 一个主要目标是 了解视紫红质的动力学;漂白时的结构变化， 与GTP酶的相互作用和磷酸化的调节， 去磷酸化 视紫红质在膜水平的研究将涉及 脱脂、变性、重折叠和重构。 一个主要目的 将是通过特定位点的结构-功能关系的研究， 基因的诱变和突变基因产物的表达。 总 已经进行了视紫红质基因的合成， 在整个基因中进行完全不受限制的诱变。 为此目的 已经引入了合适数量的独特限制性位点， 沿着基因的沿着适当位置。 棒外段结构 参与光转导的蛋白质（GT3，cGMP磷酸二酯酶， Na通道）也正在通过重组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.