UNNATURAL AMINO ACID INCORPORATION INTO PROTEINS AND QUANTIFICATION THEROF

非天然氨基酸掺入蛋白质及其定量

• 批准号: 8363805
• 负责人: Paul R Ortiz De Montellano
• 金额: $ 0.66万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363805
• 关键词: Adoption; Amino Acid Sequence; Amino Acids; Amino Acyl-tRNA Synthetases; Biochemical; Chemicals; Codon Nucleotides; Escherichia coli; Funding; Grant; Heme; In Vitro; Knowledge; Label; Ligase; Mass Spectrum Analysis; National Center for Research Resources; Peptide Sequence Determination; Positioning Attribute; Principal Investigator; Proteins; Relative (related person); Research; Research Infrastructure; Resources; Site; Source; Structure; Technology; Transfer RNA; United States National Institutes of Health; Work; cost; crosslink; in vivo

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. An approach pioneered by Prof. Peter Schultz (Scripps) allows for the site-specific incorporation of an unnatural amino acid anywhere within a protein sequence. This technology has proven useful for labeling proteins with fluorescent, 13C and 15N, and chemical-crosslinking unnatural amino acids for in vivo and in vitro structure-function studies. By using an orthogonal M. jannaschii tRNA/tRNA-synthetase pair, i.e., one where neither the tRNA nor the synthetase cross-reacts with the endogenous E.coli tRNA's or amino acyl tRNA synthetases, it is possible to introduce an unnatural amino acid residue at any position in the protein using a codon that is only recognized by the orthogonal tRNA. The Ortiz de Montellano lab acquired this labeling technology from the Schultz lab and is using it to label heme containing proteins for biophysical and biochemical studies. Information on the extent of incorporation of the unnatural amino acid and the relative expression level of the protein can severely limit the application of this technology. For that reason, we utilize the UCSF Mass Spectrometry Facility to determine the extent and site of incorporation of the unnatural amino acids into the proteins, and to determine their relative expression levels. This knowledge is not only critical for our work, but will make possible the more widespread adoption of this powerful technology.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 由Peter Schultz教授(斯克里普斯)首创的一种方法允许在蛋白质序列中的任何位置特定地结合非天然氨基酸。这项技术已被证明是有用的，用荧光标记蛋白质，13C和15N，以及化学交联的非天然氨基酸，用于体内和体外结构功能研究。通过使用正交的jannaschii tRNA/tRNA-合成酶对，即tRNA和合成酶都不与内源E.colitRNA或氨基酰基tRNA合成酶交叉反应的对，可以使用仅由正交tRNA识别的密码子在蛋白质的任何位置引入非自然的氨基酸残基。奥尔蒂斯·德·蒙特拉诺实验室从舒尔茨实验室获得了这项标记技术，并正在使用它来标记生物物理和生化研究中含有血红素的蛋白质。有关非天然氨基酸的结合程度和蛋白质的相对表达水平的信息可能会严重限制这项技术的应用。为此，我们利用加州大学旧金山分校的质谱学设备来确定非天然氨基酸在蛋白质中的结合程度和位置，并确定它们的相对表达水平。这一知识不仅对我们的工作至关重要，而且将使更广泛地采用这项强大的技术成为可能。