The conformational possibilities of three different delta-selective opioid peptides, which are DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen), DCFPE (Tyr-D-Cys-Phe-D-Pen), and DRE (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, dermenkephalin), were explored using energy calculations. Sets of low-energy conformers were obtained for each of these peptides. The sets consisted of 61 structures for DPDPE, 32 for DCFPE, and 38 for DRE, including various types of rotamers of the Tyr and Phe side-chain groups. Comparison of the geometrical shapes of the conformers was performed for these sets using topographical considerations, i.e., examination of the mutual spatial arrangement of the N-terminal alpha-amino group, and of the Tyr and Phe side-chain groups. The results obtained suggest a model for the delta-receptor-bound conformer(s) for opioid peptides. The model suggests the placement of the Phe side chain in a definite position in space corresponding to the g- rotamer of Phe for peptides containing Phe4 and to the t rotamer for peptides containing Phe. The position of the Tyr1 side chain cannot be specified so precisely. The proposed model is in a good agreement with the results of biological testing of beta-Me-Phe4-substituted DPDPE analogues that were not considered in the process of model construction.

Conformations of two cyclic analogs of angiotensin (Asp1-Arg2-Val3-Tyr4-Val/Ile5-His6-Pro7-Phe8, AT), cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT, were studied, independently employing two complementary techniques, energy calculations and NMR measurements in DMSO solution. NMR data were indicative of well-defined solution conformations for the cyclic moieties of cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT, including the phi values for the Cys3/HCys3 and Tyr4 residues, as well as the chi 1 value for the Tyr4 residue. Solution conformations for the exocyclic linear parts of both molecules cannot be described by the NMR data with the same precision. At the same time, independent energy calculations revealed the same conformations of cyclic moieties of cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT among low-energy conformers for both peptides. Moreover, the same conformations are compatible with the model of AT receptor-bound conformation (Nikiforovich & Marshall, 1993), which assumes the particular spatial arrangement of aromatic moieties of Tyr4, His6, and Phe8 residues and the C-terminal carboxyl. These conformers of cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT contain "an open turn" in the backbone of the Tyr4-Val5 residues, instead of the earlier proposed beta-like reversal, thus confirming the suggestion that the conformation(s) ensuring binding of AT analogs with specific receptors should not be described in terms of a unique backbone conformer.

A novel approach that iteratively combined the results of energy calculations and experimental data was used to generate a three-dimensional (3D) model of the photoactivated state (R*) of bovine rhodopsin (Rh). The approach started with simplified energy calculations in an effort to find a set of sterically and energetically reasonable options for transmembrane (TM) helix arrangements with all-trans-retinal. Various 3D models of TM helix packing found by computations were then compared to limited site-directed spin-label experimental data regarding the transition of the TM helices of Rh in the inactive state (R) to those in the R* state to identify the most plausible model of the TM helical bundle. At the next step, all non-TM structural elements, such as the non-TM helix 8, the N- and C-terminal fragments, and the loops connecting TM helices, were reconstructed, and after the entire R* structure had been relaxed, all other currently available additional experimental data, both mutational and spectroscopic, on the structure of the meta-II state of rhodopsin were used to validate the resulting 3D model.

The extracellular loops of G protein-coupled receptors (GPCRs) frequently contain binding sites for peptide ligands. However, the mechanism of receptor activation following ligand binding and the influence of the extracellular loops in other aspects of receptor function are poorly understood. Here we report a structure-function analysis of the first and third extracellular loops of the human C5a receptor, a GPCR that binds a 74-amino acid peptide ligand. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The first extracellular loop contains a large number of positions that cannot tolerate amino acid substitutions, especially residues within the WXFG motif found in many rhodopsin-like GPCRs, yet disruption of these residues does not alter C5a binding affinity. These results demonstrate an unanticipated role for the first extracellular loop, and the WXFG motif in particular, in ligand-mediated activation of the C5a receptor. This motif likely serves a similar role in other GPCRs. The third extracellular loop, in contrast, contains far fewer preserved residues and appears to play a less essential role in receptor activation.G protein-coupled receptors (GPCRs) 2 are the largest class of membrane-bound receptors, with more than 850 members in the human genome (1). The majority of GPCRs is grouped in the rhodopsin family by the presence of a small number of conserved amino acids in the transmembrane (TM) bundle, such as the DRY motif at the cytoplasmic end of TM3 and the NPXXY motif in TM7 (2). These receptors share a similar molecular architecture within the seven-helix bundle, which was originally represented by the ␣-carbon template of the Baldwin model (3) and later confirmed by the high resolution structure of bovine rhodopsin (4). The extracellular loops, in contrast, are more divergent, both in length and function (5). Ligands frequently bind to the extracellular loops, and the variability of the loops is not surprising when considering the range of ligands known to interact with GPCRs. For many GPCRs, especially those that bind larger peptide ligands, the mechanisms by which ligand binding stimulates G protein activation and how the extracellular loops can influence receptor activation are poorly understood. Considering that more than half of all prescribed medications target GPCRs, mostly by disrupting or mimicking ligand binding (6), a better understanding of the function of the extracellular loops is extremely important.To elucidate how GPCRs function as molecular switches to transduce signals, we study the complement factor 5a receptor (C5aR), a rhodopsin-like GPCR expressed primarily on the surface of neutrophils and other myeloid cells. C5a, a 74-amino acid peptide released during complement activation, binds to C5aR and directs neutrophil chemotaxis and the release of proteolytic enzymes and superoxide (7). C5a binds to both the transmembrane bundle and the amino terminus of C5aR (8, 9), and other binding sites in the extracellular l...

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Vinyl sulfide cyclized analogues of the octapeptide angiotensin II that are structurally related to the cyclic disulfide agonist c[Hcy(3,5)]Ang II have been prepared. The synthesis relies on the reaction of the mercapto group of a cysteine residue in position 3 with the formyl group of allysine incorporated in position 5 of angiotensin II. A mixture of the cis and the trans isomers was formed, and these were separated and isolated by RP-HPLC. Thus, the three-atom CH(2)[bond]S[bond]S element of the AT(1) receptor agonist c[Hcy(3,5)]Ang II has been displaced by a bioisosteric three-atom S[bond]CH[double bond]CH element. A comparative conformational analysis of the 13-membered ring systems of c[Hcy(3,5)]Ang II and the 13-membered cyclic vinyl sulfides with cis and trans configuration, respectively, suggested that all three systems adopted very similar low-energy conformations. This similarity was also reflected in the bioactivity. Both of the compounds that contained the ring systems encompassing the cis or trans vinyl sulfide elements between positions 3 and 5 exhibited K(i) values less than 2 nM and exerted full agonism at the AT(1) receptor. In contrast, vinyl sulfide cyclization involving the amino acid residues 5 and 7 rendered inactive compounds. The cyclic vinyl sulfides that have agonist activity were both shown to possess low-energy conformers compatible with the previously proposed 3D model for the bioactive conformation of Ang II.

Macrocyclization of a bioactive peptide with a bifunctional near-infrared fluorescent optical probe gives a compound that retained the receptor binding affinity of the peptide and the photophysical properties of the optical probe. The robust nature of the new compound provides a structural framework for optimizing the activity of bioactive molecules and for monitoring chemical or biological processes in vivo and in vitro by near-infrared optical methods.

A number of diseases have been linked to abnormal conformation of albumin, a major extracellular protein in blood. Current protein structural analysis requires pure isolated samples, thereby limiting their use for albumin analysis in blood. In this study, we report a new approach for high-throughput structure-related analysis of albumin by using the fluorescence lifetime properties of near-infrared (NIR) polymethine dyes. Based on molecular modeling, polymethine dyes are bound to two binding sites with different polarities on albumin. As a result, an NIR molecular probe exhibits two distinct lifetimes with two corresponding fluorescent fractional contributions. The distribution of fractional contributions along with individual fluorescence lifetimes represents unique parameters for characterizing albumin architecture by ratiometric analysis. After screening a small library of NIR polymethine dyes, we identified and used a polymethine dye with optimal fluorescence lifetime properties to assess structure-related differences in commercially available bovine serum albumin as model systems. The results show that changes in the lifetime of NIR dyes reflect the perturbation of the tertiary structures of albumin and that albumin prepared by different methods has slightly altered tertiary structures. Because of the reduced absorption of light by blood in the NIR region, the method developed can be used to determine structural changes in albumin in whole blood without prior isolation of the pure protein.