STRUCTURE, STABILITY AND REGULATION OF HUMAN ACIDIC FGF

人类酸性 FGF 的结构、稳定性和调控

• 批准号: 2430503
• 负责人: MICHAEL BLABER
• 金额: $ 9.4万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2430503
• 关键词: X ray crystallography; calorimetry; chemical stability; fibroblast growth factor; protein denaturation; protein structure

DESCRIPTION: This proposal describes investigations aimed at determining the role of stability and irreversible denaturation in the regulation of human acidic fibroblast growth factor (haFGF), a growth factor involved in normal embryonic development, wound healing, angiogenesis, as well as in the maintenance of some solid tumors. Like many regulatory proteins, haFGF has a short half life in vivo, which, in this case, appears to be due to generally poor thermal stability, which operates in conjunction with structural mechanisms (i.e., buried free cysteine residues) to lead to irreversible denaturation. This proposal is aimed generally at investigating protein stability as a regulatory mechanism that may be common to the regulation of short lived potent signaling proteins. A combination of methodologies will be used. Hypotheses about the structural basis of stability will be tested by the introduction of specific changes in the sequence of haFGF. X-ray crystallography will be used to determine the structural effects of these mutations. High sensitivity differential scanning calorimetry will be used to determine the effects of mutations upon thermodynamic parameters of unfolding, and upon reversible folding characteristics. Cell assays involving the stimulation of DNA synthesis in fibroblasts will be used to determine the effects of mutation upon function. Three regions of the protein are selected for mutation, based upon hypotheses of the potential origins of the protein's instability. Internal cysteine residues will be substituted by alanine to determine whether they contribute to instability, solely by promoting the irreversible phase of denaturation via disulfide formation. Lysines that form the basic cluster involved in heparin binding will be mutated to determine the role of charge repulsion within this cluster, in the absence of bound anions or heparin, in decreasing stability. Mutations will be introduced into residues surrounding the protein's central cavity, to reduce the size of this cavity, and ligands will be introduced into the central cavity, to determine the role of the cavity in protein instability.

描述：本提案描述了旨在确定 稳定性和不可逆变性在调节中的作用 人酸性成纤维细胞生长因子（haFGF）， 参与正常胚胎发育、伤口愈合、血管生成， 以及维持某些实体瘤。 像许多 作为调节蛋白，haFGF在体内具有短的半衰期，在这种情况下， 在这种情况下，似乎是由于通常较差的热稳定性， 与结构机构一起操作（即，埋入自由 半胱氨酸残基）以导致不可逆变性。 这项建议 一般旨在研究蛋白质稳定性作为调节 这可能是共同的机制，以调节短暂的有效 信号蛋白 将综合使用各种方法。 假设的 稳定性的结构基础将通过引入 haFGF序列的特定变化。X射线晶体学将 用于确定这些突变的结构效应。 高 灵敏度差示扫描量热法将用于确定 突变对解折叠热力学参数的影响，以及 可逆折叠特性。 细胞试验涉及 刺激成纤维细胞中的DNA合成将用于确定 突变对功能的影响 基于以下选择蛋白质的三个区域进行突变： 蛋白质不稳定性的潜在来源的假说。 内部半胱氨酸残基将被丙氨酸取代，以确定 他们是否有助于不稳定，仅仅是通过促进 通过二硫化物形成的不可逆变性阶段。 赖氨酸 参与肝素结合的基本簇会发生突变 为了确定电荷排斥在这个簇中的作用，在 缺乏结合阴离子或肝素，稳定性降低。 突变 将被引入蛋白质中心周围的残基中， 腔，以减小该腔的尺寸，并且将引入配体 进入中央空腔，以确定空腔在蛋白质中的作用 不稳定