Multiplicity of mechanisms of serotonin receptor signal transduction.

The serotonin (5-hydroxytryptamine, 5-HT) receptors have been divided into 7 subfamilies by convention, 6 of which include 13 different genes for G-protein-coupled receptors. Those subfamilies have been characterized by overlapping pharmacological properties, amino acid sequences, gene organization, and second messenger coupling pathways. Post-genomic modifications, such as alternative mRNA splicing or mRNA editing, creates at least 20 more G-protein-coupled 5-HT receptors, such that there are at least 30 distinct 5-HT receptors that signal through G-proteins. This review will focus on what is known about the signaling linkages of the G-protein-linked 5-HT receptors, and will highlight some fascinating new insights into 5-HT receptor signaling.

Intrahelical arrangement in the integral membrane protein rhodopsin investigated by site-specific chemical cleavage and mass spectrometry.

Site-specific cleavage on the interhelical loop I on the cytoplasmic face of rhodopsin has been observed after activation of a Cu-phenanthroline tethered cleavage reagent attached on the cytoplasmic loop IV. The characterization of the reaction products by mass spectrometry, both of the membrane-bound protein and of the CNBr-cleaved peptides, allows the site of cleavage to be determined precisely. The specific cleavage of the peptide bond between Q64 and H65 on loop I leaves the N-terminal peptide (M1-Q64) intact, confirmed by MALDI-MS detection of the two N-linked glycosyl groups near the N-terminus of rhodopsin. The limited extension of the tether side chain requires a interresidue distance between the cleavage site, Q64, and the site of ligand attachment, C316, of less than 12 A. Upon photoactivation of the receptor, no change in the cleavage pattern is observed; however, a simulated Meta II intermediate activation state indicates a much more complex cleavage pattern. The development of this cleavage method, previously used primarily as a "chemical nuclease", in combination with mass spectrometry, may provide a powerful method on membrane protein conformation studies that can be used to complement other biophysical characterizations.

NMR solution structure of a DNA dodecamer duplex containing a cis-diammineplatinum(II) d(GpG) intrastrand cross-link, the major adduct of the anticancer drug cisplatin.

The structure of the DNA duplex dodecamer, d(CCTCTGGTCTCC. GGAGACCAGAGG), containing the cisplatin d(GpG) 1,2-intrastrand cross-link at the position denoted by asterisks, was determined in solution by high-resolution 2D NMR spectroscopy and restrained molecular dynamics refinement. The cis-[Pt(NH3)2¿d(GpG-N7(1), N7(2))¿] lesion causes the adjacent guanine bases to roll toward one another by 49 degrees, leading to an overall helix bend angle of 78 degrees. These features are more exaggerated than those observed in the X-ray crystal structure determined for the same platinated duplex [Takahara et al. (1995) Nature 377, 649-652]. A common property of the solution and crystal structures is the widening and flattening of the minor groove opposite the platinum adduct, affording geometric parameters resembling those found in A-form DNA. This deformation is especially noteworthy for the solution structure because its sugar puckers are primarily those of B-form DNA. The unwinding of the helix at the site of platination is 25 degrees. The curvature and shape of the platinated duplex are remarkably similar to those observed in DNA duplexes complexed by the HMG-domain proteins SRY and LEF-1. The structure reveals how cisplatin binding alters DNA in such a manner as to facilitate HMG-domain protein recognition.

The effect of protonation on [Mn(IV)(µ2-O)] 2 complexes.

The series of complexes [Mn(IV)(X-SALPN)(µ2-O)]2, 1: X=5-OCH3; 2: X=H; 3: X=5-Cl; 4: X=3,5-diCl; 5: X=5-NO2, contain [Mn2O2](4+) cores with Mn-Mn separations of 2.7 Å. These molecules can be protonated to form [Mn(IV)(X-SALPN)(µ2-O,OH)]2 (+) in which a bridging oxide is protonated. The pKa values for the series of [Mn(IV)(X-SALPN)(µ2-O,OH)]2 (+) track linearly versus the shift in redox potential with a slope of 84 mV/pKa. This observation suggests that the [Mn2O2](4+) core can be considered as a unit in which the free energy of protonation is directly related to the ability to reduce the Mn(IV) ion. The marked sensitivity of the reduction potential to the presence of protons presents a mechanism in which an enzyme can control the oxidizing capacity of an oxo manganese cluster by the degree and timing of oxo bridge protonation.