A cleavable cross-linking reagent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-l,3'-dithiopropionate (SAED), was synthesized for the selective transfer of a coumarin fluorophore from a 'donor' protein to a position near the binding site of an interacting 'target' protein. SAED contains a terminal N-sulfosuccinimidyl ester for conjugation to the donor, a terminal photoactivatable azido-coumarin species for cross-linking with the interacting target, and a central disulfide spacer for the release of the labeled target after cleavage. To evaluate the effectiveness of this labeling reagent, soybean trypsin inhibitor (STI) was derivatized (~0 . 5 mol/mol) with SAED and then photolyzed in the presence of trypsin. A single fluorescent cross-linked species (6 -7 mol% of total STI) was observed by SDSjPAGE and, after reductive cleavage, was shown to be a 1 : 1 STItrypsin complex. This complex was not detected without photolysis or with an inactivated crosslinker. Importantly, complex formation was inhibited by an excess of unmodified STI and prevented by substitution of a non-interacting protein for trypsin. Cleavage of the cross-linked complex revealed that the trypsin, but not the STI, was fluorescent; the uncomplexed trypsin fraction remained unlabeled. These results demonstrated the specificity of the labeling of trypsin by fluorescent-transfer cross-linking with SAED. An efficiency of about 15% for this cross-linking mediated labeling of trypsin was calculated. The short cross-linking span of SAED ( 5 1.8 nm) strictly limited the labeling to the vicinity of the contact region of trypsin with STI. Thus, this novel cross-linker permits the region-specific targeting of a fluorophore near a functionally important binding site.Chemical cross-linking reagents have been used for the determination of nearest-neighbor analysis, molecular interactions and orientations, as well as three-dimensional protein structures (Wang and Richards, 1974;Peters and Richards, 1977;Das and Fox, 1979). For these applications, the efficiency and the specificity of the coupling reaction has been increased by the development of photoaffinity heterobifunctional cross-linkers (Huang and Richards, 1977; Ji, 1979; Jaffe et al., 1980). Such reagents are first coupled to one of the components to be cross-linked while their other functional group remains chemically inert until photoactivation is initiated in the presence of the interacting component. With this controlled and sequential coupling process, self-conjugation and polymerization are kept to a minimum. More importantly, photoaffinity reagents such as aryl azides are activated into nitrenes which react extremely rapidly with numerous neighboring functional groups (Bayley and Staros, 1984).
The results of this study show that classic olefination chemistry can be adapted to a solid-phase format for parallel synthesis of analog libraries. Although yields varied for the individual analogs, sufficient quantity of pure material was obtained directly from the resin for structural characterization and biological evaluation. This study further validates solid-phase organic synthesis as a useful approach for rapid parallel-manifold library synthesis to augment both lead compound discovery and optimization.
The study findings provide information about early career paths of investigators at top-tier research institutions. In addition, the views of this successful cohort serve to inform the current dialogue and questions that remain about the future health of biomedical research and education in the United States. Educators, prospective and current students, and members of the policy community may find it useful to consider these findings and the questions they raise, some of which the authors present.
The cell membrane is a complex mixture of several classes of biomolecules but amino acids and lipids are the main constituents. For this reason we are establishing a data base of transmembrane proteins with the intent of using the data base to identify interfacial peptide sequences useful for studying protein-lipid interactions at membrane interfaces. Our present intention is to characterize transmembrane peptides and amino acids found near the membrane interface. A data base containing only signal peptides is available (G. von Heijne, Prot. Seq. Data Anal. 1:41-42, 1987).
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