STRUCTURE, STABILITY AND REGULATION OF HUMAN ACIDIC FGF

描述：本提案描述了旨在确定 稳定性和不可逆变性在调控中的作用 人酸性成纤维细胞生长因子是一种生长因子 参与正常的胚胎发育，伤口愈合，血管生成， 以及对一些实体肿瘤的维护。像许多人一样 调节蛋白，haFGF在体内有很短的半衰期，在这个过程中 情况下，似乎是由于普遍较差的热稳定性，这 与结构机制(即，自由埋藏)协同工作 半胱氨酸残基)导致不可逆变性。这项建议 通常旨在研究蛋白质的稳定性作为一种调节 可能是调节短命强效的共同机制 信号蛋白。 将结合使用各种方法。关于生物多样性的假说 稳定性的结构基础将通过引入 Ha成纤维细胞生长因子序列的特异性变化。X射线结晶学将 用来确定这些突变的结构效应。高 将使用灵敏度差示扫描量热法来确定 突变对去折叠热力学参数的影响，以及 根据可逆的折叠特性。细胞分析涉及到 对成纤维细胞DNA合成的刺激将被用来确定 突变对功能的影响。 选择蛋白质的三个区域进行突变，基于 蛋白质不稳定的潜在来源的假说。 体内的半胱氨酸残基将被丙氨酸取代以确定 它们是否会导致不稳定，仅仅是通过促进 通过形成二硫化物而发生的不可逆变性阶段。赖氨酸 形成与肝素结合有关的碱性簇的基因将发生突变 为了确定电荷斥力在这个星系团中的作用， 缺乏结合的阴离子或肝素，稳定性下降。突变 将被引入到蛋白质中心周围的残基中 空腔，以减小这个空腔的大小，并将配体引入 进入中央空腔，以确定空腔在蛋白质中的作用 不稳定。

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.