MOLECULAR RECOGNITION IN NUCLEAR PROTEIN TRANSPORT

核蛋白进口是一种精心策划和监管的 许多蛋白质，特别是激活基因的转录因子 选择性地，仅在对特定信号作出反应时才进入核内。 细胞分裂、免疫反应、分化、发育、病毒感染 其他重要的进程与进口进程密切相关。 核定位信号(NLSS)，它指导蛋白质的核进入， 是由特定的异源二聚体受体识别的短肽基序 核黄素((/())。该提案描述了一种开发 光调控核粘附素配体作为控制蛋白质的工具 有光的活体内定位。 新型的、可光化学调节的氨基酸和羧酸将是 用来产生天然信号的突变体，这些信号与核粘附素结合在一起 光调控方式。试剂是基于偶氮苯发色团的 分成两组：光弹氨基酸，它能扩张和收缩 多肽主链和α，α-二氟膦甲基偶氮苯 旨在模拟可逆的磷酸化反应对光的反应。 这些试剂将被应用于一种潜在的系统化总体战略 以NLSS和近端调节区为靶点的突变；主要 靶点包括SV40T抗原NLS和双链核纤溶酶NLS。 诱变将通过在固体上进行高效的并行合成来实现 相，候选者将在固相上进行筛选以进行差异化 与核粘附素结合，对光作出反应。有待检验的具体假设 两个肽段对受体的螯合能力可以是 由光弹氨基酸控制，并改变了一个 光诱导的磷酸同工酶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者将结合到 蛋白质携带者。这些蛋白质将被引入通透性哺乳动物体内。 细胞，光对其细胞内定位的影响将是 下定决心。据预计，信号-蛋白质偶联物，引入到 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.