CORE--Solution properties of alternatively spliced proteins / Core 1

CORE--可变剪接蛋白质的溶液特性/核心 1

• 批准号: 6689855
• 负责人: PHILIP N BRYAN
• 金额: $ 5.1万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6689855
• 关键词: biomedical facility; chromatography; circular dichroism; conformation; light scattering; mass spectrometry; nuclear magnetic resonance spectroscopy; protein folding; protein structure function; ultracentrifugation

This is a new core component of the program project 'From Genomic Sequences to Protein Structure and function'. The goal is to determine the solution properties of proteins selected for structural studies. It is expected that many of the human proteins studied in the next phase will require encouragement to fold into their functional conformation, and that a number of the alternatively spliced proteins may not have a folded functional structure. Although it is a small core, it is essential for success, as it will ensure that proper emphasis is placed on determining the solution properties of our proteins. The Pl's extensive experience in studying protein folding, and dealing with solution properties of proteins will be of value throughout the project. A set of standard biophysical techniques wilt be used: (1) Circular dichroism, to obtain an initial indication of the folding state of each protein. (2) One and two-dimensional NMR spectra, to provide precise information of the extent and level of ordering of each protein. (3). MALDI-TOF mass spectroscopy, to provide precise molecular weight and purity information. (4). Dynamic Light Scattering, for initial identification of large aggregates that might interfere with crystallization, and for establishing conditions in which the protein is monodisperse. (5) Size exclusion chromatography, used in addition to Dynamic Light Scattering, to assess the homogeneity, aggregation, and oligomeric state of the sample. (6). Ultracentrifugation, to determine accurately the oligomeric state of each suitable protein. It is expected that in some cases, different alternative splice protein versions will have different oligomeric states.

这是程序项目“从基因组序列到蛋白质结构和功能”的新核心组成部分。目的是确定选择用于结构研究的蛋白质的溶液特性。预计在下一阶段研究的许多人蛋白将需要鼓励将其折叠成其功能构象，并且许多剪接的蛋白质可能没有折叠的功能结构。尽管它是一个很小的核心，但它对于成功是必不可少的，因为它将确保适当强调确定蛋白质的溶液特性。 PL在研究蛋白质折叠方面的丰富经验以及在整个项目中都具有价值。使用一组标准的生物物理技术： （1）圆二色性，以获得每种蛋白质折叠状态的初始指示。 （2）一维NMR光谱，以提供每种蛋白质排序程度和水平的精确信息。 （3）。 MALDI-TOF质谱，以提供精确的分子量和纯度信息。 （4）。动态光散射，用于初始鉴定可能干扰的大骨料 结晶，并为建立蛋白质是单分散的条件。 （5）除了动态光散射外，还用于评估样品的均匀性，聚集和寡聚状态。 （6）。超速离心，以准确确定每个合适蛋白质的低聚状态。预计在某些情况下，不同的替代剪接蛋白版本将具有不同的寡聚状态。