MOLECULAR RECOGNITION IN NUCLEAR PROTEIN TRANSPORT

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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.