OPTICAL STUDIES OF PROTEIN STRUCTURE AND FUNCTION

蛋白质结构和功能的光学研究

• 批准号: 3272024
• 负责人: LUBERT STRYER
• 金额: $ 21.63万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-01-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3272024
• 关键词: X ray crystallography; biological signal transduction; calcium; calcium binding protein; calmodulin; calmodulin dependent protein kinase; chemical binding; chemical kinetics; circular dichroism; conformation; enzyme activity; fluorescence microscopy; fluorescence spectrometry; fluorescent dye /probe; fluorimetry; genetic library; guanylate cyclase; laser spectrometry; method development; microcalorimetry; molecular site; mutant; nuclear magnetic resonance spectroscopy; phosphorylation; protein folding; protein structure; recombinant DNA; site directed mutagenesis; stop flow technique; ytterbium

The overall goal of this research program is to develop novel fluorescence techniques and to use them in concert with other experimental approaches to elucidate the structure and dynamics of calcium-sensing proteins. Recoverin, a recently discovered member of the EF hand superfamily of calcium-binding proteins, plays a key role in vision by activating guanylate cyclase when the cytosolic calcium level is lowered following illumination. The presence of antibodies to recoverin in patients with cancer-associated retinopathy, an autoimmune retinal degenerative disease induced by peripheral tumors, suggests that homologs of recoverin are widely distributed. We have recently cloned recoverin cDNA and obtained high level expression of functional protein in E. coli. The following biophysical, biochemical, and molecular genetic studies will be carried out: (1) The kinetics, thermodynamics, and cooperativity of calcium binding will be determined fluorimetrically and microcalorimetrically. (2) Calcium-induced conformational transitions will be detected by picosecond emission anisotropy measurements, fluorescence energy transfer, and low- angle x-ray scattering. (3) Large quantities of recombinant recoverin will be produced to obtain crystals for x-ray analysis. (4) Site-specific mutants will be generated and analyzed to identify residues that are critical for calcium binding, conformational switching, and target binding. (5) The folding of recoverin will be investigated by fluorescence spectroscopy and other physical methods to establish the temporal sequence of compaction and acquisition of secondary and tertiary structure. (6) cDNA libraries and expression libraries will be screened for homologs of recoverin outside the retina. Another major aim of this research is to use fluorescence spectroscopy to elucidate how type II multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is activated by Ca2+- calmodulin and by autophosphorylation. CaM kinase, a major brain protein, regulates neurotransmitter synthesis and release, and also plays key roles in peripheral tissues, where it controls membrane channels. The proposed research will give insight into the mechanism of transduction of calcium signals and provide a basis for designing therapeutic agents that selectively modify particular pathways. We will also continue to develop new methods for high-sensitivity fluorescence detection. Picosecond laser excitation and microchannel plate detection will be used to enhance rejection of background scattering. Fluorescence detection of single molecules such as phycoerythrin-labeled antibodies and DNA probes will open new vistas in medical diagnostics.

这项研究计划的总体目标是开发新的荧光 技术，并使用它们与其他实验方法， 阐明钙敏感蛋白的结构和动力学。 恢复蛋白，最近发现的EF手超家族的成员， 钙结合蛋白，通过激活 鸟苷酸环化酶，当细胞溶质钙水平降低， 照明。 患者中恢复素抗体的存在 癌症相关性视网膜病变，一种自身免疫性视网膜变性疾病 外周肿瘤诱导，表明恢复素的同源物是 分布广泛。 我们最近克隆了recoverin cDNA， 在E.杆菌 以下 将进行生物物理、生物化学和分子遗传学研究 结果：（1）钙离子的动力学、热力学和协同效应 将通过荧光法和微量热法测定结合。 （二） 钙诱导的构象转变将被皮秒探测到 发射各向异性测量，荧光能量转移，和低- 角X射线散射 (3)大量的重组回收将 以获得用于X射线分析的晶体。 (4)位点特异 将产生突变体并分析以鉴定 对于钙结合、构象转换和靶结合至关重要。 (5)将通过荧光法研究recoverin的折叠 光谱学和其他物理方法来建立时间序列 压实和二级和三级结构的获取。 （六） 将筛选cDNA文库和表达文库中的同源物， 视网膜外的恢复。 这项研究的另一个主要目的是使用 荧光光谱来阐明II型多功能 Ca 2 +/钙调素依赖性蛋白激酶（CaM激酶）被Ca 2 +-激活， 钙调素和自身磷酸化。 钙调素激酶，一种主要的脑蛋白， 调节神经递质的合成和释放， 在外周组织中，它控制着膜通道。 拟议 研究将使人们了解钙离子的传导机制， 信号并为设计治疗剂提供基础， 选择性地改变特定的途径。 我们还将继续发展 高灵敏度荧光检测的新方法。 皮秒激光 激发和微通道板检测将用于增强 背景散射的抑制。 荧光检测单个 藻蓝蛋白标记的抗体和DNA探针等分子会打开 医学诊断的新前景