Structural Investigations of Metalloprotein Metal Sites

描述（由申请人提供）：本提案的长期目标是详细描述生物系统中锌的结构和功能特性。锌是金属蛋白中最常见的金属，也是已知的唯一一种主要酶催化所必需的金属。目前已分离出数百种锌蛋白，并在蛋白质序列中鉴定出数千个潜在的锌结合位点。锌水平的不平衡或其运输或调节的错误会对健康产生深远的影响。锌体内平衡失调与多种疾病有关，包括肌萎缩侧索硬化症、阿尔茨海默病和海绵状脑病；作为突触传递的调节剂，锌被认为在大脑的正常运作中起着关键作用。尽管它很重要，但由于研究这种光谱上“沉默”的金属的困难，关于生物锌位点的信息相对较少。x射线吸收光谱是为数不多的能够提供非晶体材料结构信息的方法之一，将用于确定一类新型含锌烷基转移酶中锌的结构，并表征金属立体化学偏好和蛋白质结构在确定金属结合位点结构中的相对重要性。对于后一项工作，将比较一系列光谱沉默金属离子(Cu(l), Ag(l), Cd(ll), Hg(ll), Pb（ll）和As（ll)，以及Zn(ll)）的结构。作为“传统”x射线光谱的补充，将开发使用高分辨率x射线发射光谱和锌l边x射线光谱的新工具。原位x射线微探针成像和微光谱学将用于表征完整生物组织中的金属位点，而新开发的毛细管电泳/x射线荧光工具将用于确定金属蛋白中的金属负载，特别是蛋白质超氧化物歧化酶。

DESCRIPTION (provided by applicant): The long term objective of this proposal is to characterize in detail the structural and functional properties of zinc in biological systems. Zinc is the most common metal found in metalloproteins and is the only metal that is known to be required for every major class of enzyme catalysis. Hundreds of zinc proteins have been isolated and thousands of potential zinc binding sites have been identified in protein sequences. Imbalances in the levels of zinc or errors in its transport or regulation can have profound health consequence. Malfunctions in zinc homeostasis have been implicated in a wide range of diseases, including amyotrophic lateral sclerosis, Alzheimer's disease, and spongiform encephalopathies; as a modulator of synaptic transmission, zinc has been suggested to play a critical role in the proper functioning of the brain. Despite its importance, there is relatively little information available about biological zinc sites due to the difficulty of studying this spectroscopically "silent" metal. X-ray absorption spectroscopy, one of the few methods able to provide structural information for non-crystalline materials, will be used to determine the structures of zinc in a novel class of zinc-containing alkyl transfer enzymes and to characterize the relative importance of metal stereochemical preference and protein structure in defining the structure of a metal binding site. For the latter work, structures will be compared for a series of spectroscopically silent metal ions (Cu(l), Ag(l), Cd(ll), Hg(ll), Pb(ll), and As(lll), in addition to Zn(ll)). As a complement to "conventional" x-ray spectroscopy, new tools will be developed using high-resolution x-ray emission spectroscopy and zinc L-edge x-ray spectroscopy. In situ x-ray microprobe imaging and microspectroscopy will be used to characterize metal sites in intact biological tissue, and the newly developed tool of capillary electrophoresis/x-ray fluorescence will be used to determine the metal loading in metalloproteins, with particularly emphasis on the protein superoxide dismutase.