APPLICATION OF MASS SPECTROMETRY TO STRUCTURAL BIOLOGY

质谱在结构生物学中的应用

• 批准号: 6106708
• 负责人: Kenneth Tomer
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6106708
• 关键词: acylation; apolipoproteins; biomedical equipment development; blood lipoprotein biosynthesis; cholesterol; cysteine; electrospray ionization mass spectrometry; fatty acids; high density lipoproteins; insulin receptor; intermolecular interaction; intracellular transport; liver cells; mass spectrometry; polymers; posttranslational modifications; protein structure; structural biology; tissue /cell culture

Summary of Work: Recent instrumental developments in mass spectrometry, such as matrix-assisted laser desorption and electrospray ionization, have enabled mass spectrometrists to investigate biological molecules with significantly higher Mr than previously possible. The combination of these techniques with chemical processing of large biopolymers such as proteins now enables the use of mass spectrometry to play a significant role in the realm of structural biology. In this respect, structural biology not only refers to the determination of the primary sequence and sites of post-translational modifications, but also to probing the tertiary structure of molecules and complexes. We are currently working on several projects. Short descriptions of some of these follows: ? The first project is to develop the capability of probing non-covalent complexes between proteins and DNA, using the yeast transcription factor GCN4. These proteins contain a basic DNA-binding domain and a leucine zipper dimerization domain, and the dimers specifically bind dsDNA to form a tetramolecular noncovalent complex. Using ESI, we have observed, for the first time, such specific tetramolecular complexes bv MS. We hope, eventually, to include DNA:protein footprinting experiments into this project. ? A second project is to identify the residues on the human p53 tumor suppressor protein post-translationally phosphorylated in response to radiation induced DNA damage (in collaboration with Merrick, LMC). We have found 10 different phosphorylated isoforms of p53 in human mammary epithelial cells and recombinant p53. We hypothesize that the specific phosphorylation pattern of p53 determines the decision for mitotic arrest or apoptosis in response to DNA damage. Using MS/MS we have so far unequivocally identified one phosphorylation site. ? Structure determination of the catalytic core of protein phosphatase 5 (a serine/threonine phosphatase which may mediate the effect of some lipids on ion channel activity) that is released from a regulatory region upon subtilisin digestion and how this sequence differs from the similar core released by trypsin. The sequences of the subtilisin and trypsin cleaved fragments have been determined. This project is in collaboration with the NIEHS/LST and Purdue Univ. groups. ? Structure determination of the protein residues involved in Schiff base formation in the intermediate formed in base excision repair by Pol beta (Collaboration with S. Wilson, LSB) ? Identification of phosphorylated residues on TTP, an RNA binding protein, and identification of residues on this protein that are crosslinked with RNA using a photoinducible crosslinking reagent (with P. Blackshear, DIR).

工作总结：最近的仪器