Nicotinic acetylcholine (ACh) receptor (nAChR) agonists are potential therapeutic agents for neurological dysfunction. In the present study, the homopentameric mollusk ACh binding protein (AChBP), used as a surrogate for the extracellular ligand-binding domain of the nAChR, was specifically derivatized by the highly potent agonist azidoepibatidine (AzEPI) prepared as a photoaffinity probe and radioligand. One EPI-nitrene photoactivated molecule was incorporated in each subunit interface binding site based on analysis of the intact derivatized protein. Tryptic fragments of the modified AChBP were analyzed by collision-induced dissociation and Edman sequencing of radiolabeled peptides. Each specific EPI-nitrene-modified site involved either Tyr195 of loop C on the principal or (+)-face or Met116 of loop E on the complementary or (-)-face. The two derivatization sites were observed in similar frequency, providing evidence of the reactivity of the azido/nitrene probe substituent and close proximity to both residues. [3H]AzEPI binds to the alpha4beta2 nAChR at a single high-affinity site and photoaffinity-labels only the alpha4 subunit, presumably modifying Tyr225 spatially corresponding to Tyr195 of AChBP. Phe137 of the beta2 nAChR subunit, equivalent to Met116 of AChBP, conceivably lacks sufficient reactivity with the nitrene generated from the probe. The present photoaffinity labeling in a physiologically relevant condition combined with the crystal structure of AChBP allows development of precise structural models for the AzEPI interactions with AChBP and alpha4beta2 nAChR. These findings enabled us to use AChBP as a structural surrogate to define the nAChR agonist site.

The Ni(II) complex of the tripeptide NH 2 -glycine-glycine-histidine-COOH (GGH) mediates efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid (MMPP). Here we demonstrate that GGH fused to the amino terminus of a protein can still support cross-linking. The tripeptide was expressed at the amino terminus of ecotin, a dimeric macromolecular serine protease inhibitor found in the periplasm of Escherichia coli. In the presence of Ni(OAc) 2 and MMPP, GGH-ecotin is cross-linked to give a species that has an apparent molecular mass of a GGHecotin dimer with no observable protein degradation. The cross-linking reaction occurs between two ecotin proteins in a dimer complex. Furthermore, GGH-ecotin can be cross-linked to a serine protease target, trypsin, and the reaction is specific for proteins that interact with ecotin. The cross-linking reaction has been carried out on small peptides, and the reaction products have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The target of the reaction is tyrosine, and the product is bityrosyl cross-links. The yield of the cross-linking is on the order of 15%. However, the reaction efficiency can be increased 4-fold by a single amino acid substitution in the carboxy terminus of ecotin that places an engineered tyrosine within 5 Å of a naturally occurring tyrosine. This cross-linking methodology allows for the protein cross-linking reagent to be encoded for at the DNA level, thus circumventing the need for posttranslational modification.

The relative rates for the gas-phase dissociation RX(+) --> R(+) + X degrees of five (4-Y-substituted benzyl)dimethysulfoniums (Y = MeO, Me, H, Cl, and NO(2)) and 24 (4-Y-substituted benzyl)-3'-Z-pyridiniums (complete series for Z = CN, Cl, CONH(2), and H, and 4-methoxy- and 4-nitrobenzyls for Z = F and CH(3)CO) were measured using liquid secondary ion mass spectrometry. The Hammett plot (vs deltaDeltaG degrees or sigma(+)) is linear for the sulfoniums, but plots for the four pyridinium series have a drastic break between the 4-Cl and 4-NO(2) substrates. Brønsted-like plots for the pyridiniums show a strong leaving group effect only for 4-nitrobenzyls. An analysis of these linear free energy relations with supporting evidence from semiempirical computations suggests that collisionally activated pyridinium substrates dissociate by two pathways, direct dissociation and through an ion-neutral complex intermediate. Comparison of these results with results for the solution reactions of some of these compounds shows that the mechanism is different in the gas and solution phases. Sufficient experimental data are not available to assign a mechanism for dissociation to the sulfonium series, but computational results show characteristics of a direct dissociative mechanism.