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“从基因组序列到蛋白质结构和功能”项目的新核心组成部分。目标是确定选择用于结构研究的蛋白质的溶液性质。预计在下一阶段研究的许多人类蛋白质将需要鼓励折叠成它们的功能构象，并且许多选择性剪接的蛋白质可能不具有折叠的功能结构。虽然它是一个小的核心，但它是成功的关键，因为它将确保适当的重点放在确定我们蛋白质的溶液性质上。Pl在研究蛋白质折叠和处理蛋白质溶液性质方面的丰富经验将在整个项目中发挥重要作用。将使用一套标准的生物物理技术： (1)圆二色性，以获得每个蛋白质折叠状态的初始指示。 (2)一维和二维核磁共振谱，提供每种蛋白质有序化程度和水平的精确信息。 （三）、MALDI-TOF质谱，提供精确的分子量和纯度信息。 （四）、动态光散射，用于初始识别可能干扰 结晶，以及建立蛋白质是单分散的条件。 (5)分子排阻色谱法，除动态光散射法外，还用于评估样品的均匀性、聚集和寡聚状态。 （六）、超离心，以准确测定每种合适蛋白质的寡聚状态。预期在某些情况下，不同的可变剪接蛋白形式将具有不同的寡聚状态。