MOLECULAR RECOGNITION IN NUCLEAR PROTEIN TRANSPORT

核蛋白输入是一个精心策划和调节的过程， 许多蛋白质，特别是激活基因的转录因子 只有在特定信号的作用下才有选择性地进入细胞核。 细胞分裂、免疫反应、分化、发育、病毒感染 和其他重要过程与进口过程密切相关。 核定位信号（NLS），指导蛋白质进入核， 是由特异性异二聚体受体识别的短肽基序 （karyopherin（）.该提案描述了一种方法， 作为控制蛋白质的工具的光调节的核转运蛋白配体 在体内用光定位。 新的，光化学可调节的氨基酸和羧酸将是 用于产生天然信号的突变体，所述天然信号结合细胞核转运蛋白， 光调节方式。这些试剂基于偶氮苯发色团 分为两类：光弹性氨基酸， 肽骨架和α，α-二氟-膦酰基甲基偶氮苯， 旨在模拟响应于光的可逆磷酸化。 这些试剂将应用于一种潜在的系统性的一般策略， 靶向天然NLS和近端调控区的诱变;主要的 靶包括SV 40 T抗原NLS和二分核质蛋白NLS。 诱变将通过在固体上的高效平行合成来实施。 阶段，候选人将在固相上进行筛选， 对光有反应时与核转运蛋白结合。待检验的具体假设 两个肽结构域螯合受体的能力可以 用光弹性氨基酸控制，改变了一个 光诱导的磷酸盐电子等排体可以改变细胞的受体结合特性， 信号. 显示光敏受体结合的候选物将缀合至 蛋白质载体将蛋白质引入透化的哺乳动物细胞中， 细胞，光对其细胞内定位的影响将是 测定预期引入到细胞中的信号蛋白缀合物， 活细胞，会有光依赖的定位，可以用来 控制细胞过程，如基因转录。

Nuclear protein import is a carefully orchestrated and regulated process; many proteins, notably transcription factors that activate genes selectively, are moved into the nucleus only in response to specific signals. Cell division, immune response, differentiation, development, viral infection and other important processes are intimately tied to the import process. Nuclear localization signals (NLSs), which direct nuclear entry of proteins, are short peptide motifs recognized by a specific, heterodimeric receptor (karyopherin ((/(). This proposal describes an approach to developing photo-regulated karyopherin ligands as tools for controlling protein localization in vivo with light. Novel, photochemically regulatable amino acids and carboxylic acids will be employed to generate mutants of native signals that bind karyopherin in a photo-regulated manner. The reagents are based on the azobenzene chromophore and fall into two groups: photoelastic amino acids that expand and contract the peptide backbone, and alpha, alpha-difluoro-phosphonomethyl azobenzenes which are intended to mimic reversible phosphorylation in response to light. The reagents will be applied in a potentially general strategy of systematic mutagenesis that targets native NLSs and proximal regulatory regions; principal targets include the SV40 T antigen NLS and the bipartite nucleoplasmin NLS. Mutagenesis will be implemented by efficient, parallel synthesis on the solid phase, and candidates will be screened on the solid phase for differential binding to karyopherin in response to light. Specific hypotheses to be tested are that the ability of two peptide domains to chelate the receptor can be controlled with photo-elastic amino acids, and that altered placement of a phosphate isostere by light can alter receptor-binding properties of the signals. Candidates displaying photosensitive receptor binding will be conjugated to protein carriers. The proteins will be introduced into permeabilized mammalian cells, and the effect of light on their intracellular localization will be determined. It is anticipated that signal-protein conjugates, introduced into living cells, would have light-dependent localization that could be used to control cellular processes such as gene transcription.