OPTICAL STUDIES OF PROTEIN STRUCTURE AND FUNCTION

这项研究计划的总体目标是开发新的荧光 技术，并将它们与其他实验方法配合使用，以 阐明钙敏感蛋白的结构和动力学。 Recoverin，最近发现的EF Hand超家族成员 钙结合蛋白，通过激活在视觉中起关键作用 鸟苷环化酶在细胞内钙水平降低时的作用 照明。慢性粒细胞白血病患者血清中抗恢复素抗体的检测 癌症相关性视网膜病变--一种自身免疫性视网膜退行性疾病 由外周肿瘤诱导，表明恢复素的同源物是 分布广泛。我们最近克隆了恢复素基因，并获得了 功能蛋白在大肠杆菌中的高效表达。以下是 将进行生物物理、生化和分子遗传学研究 Out：(1)钙的动力学、热力学和协同性 结合作用将用荧光法和微量热法测定。(2) 钙诱导的构象转变将在皮秒内被检测到 发射各向异性测量、荧光能量转移和低密度 角X射线散射。(3)大量重组恢复素 被生产出来，以获得用于X射线分析的晶体。(4)特定地点 将产生并分析突变体，以确定哪些残基 对钙结合、构象转换和靶结合至关重要。 (5)用荧光法研究恢复素的折叠。 光谱学和其他物理方法来建立时间序列 压实和获得二级和三级结构。(6) 将筛选cDNA文库和表达文库中的同源物 在视网膜外恢复。这项研究的另一个主要目的是使用 荧光光谱学解释II型多功能 钙/钙调蛋白依赖的蛋白激酶(CaM)被钙激活- 钙调蛋白和自磷酸化。CaM激酶，一种主要的大脑蛋白质， 调节神经递质的合成和释放，也起着关键作用 在外周组织中，它控制着膜通道。建议数 研究将深入了解钙离子的转导机制 信号，并为设计治疗剂提供基础 有选择地修改特定的路径。我们也将继续发展 高灵敏度荧光检测的新方法。皮秒激光 将使用激励和微通道板检测来增强 背景散射抑制。单次荧光检测 藻红蛋白标记的抗体和DNA探针等分子将打开 医学诊断学的新前景。

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.