Thrombostatin FM compounds: direct thrombin inhibitors - mechanism of action in vitro and in vivo.

BACKGROUND: Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D-isomers and unusual amino acids were prepared based upon the stable angiotensin converting enzyme breakdown product of bradykinin - RPPGF. METHODS AND RESULTS: These peptides are direct thrombin inhibitors prolonging the thrombin clotting time, activated partial thromboplastin time, and prothrombin time at >or=0.78, 1.6, and 1.6 microm, respectively. They competitively inhibit alpha-thrombin-induced cleavage of a chromogenic substrate at 4.4-8.2 microm. They do not significantly inhibit plasma kallikrein, factor (F) XIIa, FXIa, FIXa, FVIIa-TF, FXa, plasmin or cathepsin G. One form, FM19 [rOicPaF(p-Me)], blocks alpha-thrombin-induced calcium flux in fibroblasts with an IC(50) of 6.9 +/- 1.2 microm. FM19 achieved 100% inhibition of threshold alpha- or gamma-thrombin-induced platelet aggregation at 8.4 +/- 4.7 microm and 16 +/- 4 microm, respectively. The crystal structure of thrombin in complex with FM19 shows that the N-terminal D-Arg retrobinds into the S1 pocket, its second residue Oic interacts with His-57, Tyr-60a and Trp-60d, and its C-terminal p-methyl Phe engages thrombin's aryl binding site composed of Ile-174, Trp-215, and Leu-99. When administered intraperitoneal, intraduodenal, or orally to mice, FM19 prolongs thrombin clotting times and delays carotid artery thrombosis. CONCLUSION: FM19, a low affinity reversible direct thrombin inhibitor, might be useful as an add-on agent to address an unmet need in platelet inhibition in acute coronary syndromes in diabetics and others who with all current antiplatelet therapy still have reactive platelets.

Design of high affinity cyclic pentapeptide ligands for kappa-opioid receptors.

Using results from our previously reported cyclic opioid peptide series and reliable models for mu-, delta-, and kappa-opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr-C[D-Cys-Phe-Phe-X]-NH2, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = D- or L-Cys; or D- or L-penicillamine (Pen; beta,beta-dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = D- or L-Cys display KOR affinities in the low nanomolar range, demonstrating that a 'DPDPE-like' tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8, Tyr-C[D-Cys-Phe-Phe-Cys]-NH2-cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm) with more than 100-fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.

Roles of residues 3 and 4 in cyclic tetrapeptide ligand recognition by the kappa-opioid receptor.

A series of cyclic, disulfide- or dithioether-containing tetrapeptides based on previously reported potent mu- and delta-selective analogs has been explored with the aim of improving their poor affinity to the kappa-opioid receptor. Specifically targeted were modifications of tetrapeptide residues 3 and 4, as they presumably interact with residues from transmembrane helices 6 and 7 and extracellular loop 3 that differ among the three receptors. Accordingly, tetrapeptides were synthesized with Phe(3) replaced by aliphatic (Gly, Ala, Aib, Cha), basic (Lys, Arg, homo-Arg), or aromatic sides chains (Trp, Tyr, p-NH(2)Phe), and with d-Pen(4) replaced by d-Cys(4), and binding affinities to stably expressed mu-, delta-, and kappa-receptors were determined. In general, the resulting analogs failed to exhibit appreciable affinity for the kappa-receptor, with the exception of the tetrapeptide Tyr-c[d-Cys-Phe-d-Cys]-NH(2), cyclized via a disulfide bond, which demonstrated high binding affinity toward all opioid receptors (Ki(mu) = 1.26 nm, Ki(delta) = 16.1 nm, Ki(kappa) = 38.7 nm). Modeling of the kappa-receptor/ligand complex in the active state reveals that the receptor-binding pocket for residues 3 and 4 of the tetrapeptide ligands is smaller than that in the mu-receptor and requires, for optimal fit, that the tripeptide cycle of the ligand assume a higher energy conformation. The magnitude of this energy penalty depends on the nature of the fourth residue of the peptide (d-Pen or d-Cys) and correlates well with the observed kappa-receptor binding affinity.

Quantification of helix-helix binding affinities in micelles and lipid bilayers.

A theoretical approach for estimating association free energies of alpha-helices in nonpolar media has been developed. The parameters of energy functions have been derived from DeltaDeltaG values of mutants in water-soluble proteins and partitioning of organic solutes between water and nonpolar solvents. The proposed approach was verified successfully against three sets of published data: (1) dissociation constants of alpha-helical oligomers formed by 27 hydrophobic peptides; (2) stabilities of 22 bacteriorhodopsin mutants, and (3) protein-ligand binding affinities in aqueous solution. It has been found that coalescence of helices is driven exclusively by van der Waals interactions and H-bonds, whereas the principal destabilizing contributions are represented by side-chain conformational entropy and transfer energy of atoms from a detergent or lipid to the protein interior. Electrostatic interactions of alpha-helices were relatively weak but important for reproducing the experimental data. Immobilization free energy, which originates from restricting rotational and translational rigid-body movements of molecules during their association, was found to be less than 1 kcal/mole. The energetics of amino acid substitutions in bacteriorhodopsin was complicated by specific binding of lipid and water molecules to cavities created in certain mutants.

Structure-based design, synthesis, and pharmacologic evaluation of peptide RGS4 inhibitors.

Regulators of G-protein signaling (RGS) proteins form a multifunctional signaling family. A key role of RGS proteins is binding to the G-protein Galpha-subunit and acting as GTPase-activating proteins (GAPs), thereby rapidly terminating G protein-coupled receptor (GPCR) signaling. Using the published RGS4-Gialpha1 X-ray structure we have designed and synthesized a series of cyclic peptides, modeled on the Gialpha Switch I region, that inhibit RGS4 GAP activity. These compounds should prove useful for elucidating RGS-mediated activity and serve as a starting point for the development of a novel class of therapeutic agent.

Development and validation of opioid ligand-receptor interaction models: the structural basis of mu vs delta selectivity.

Opioid receptor binding conformations for two structurally related, conformationally constrained tetrapeptides, JOM-6 ( micro receptor selective) and JOM-13 (delta receptor selective), were deduced using conformational analysis of these ligands and analogs with additional conformational restrictions. Docking of these ligands in their binding conformations to opioid receptor structural models, based upon the published rhodopsin X-ray structure, implicates specific structural features of the micro and delta receptor ligand binding sites as forming the basis for the micro selectivity of JOM-6 and the delta selectivity of JOM-13. In particular, the presence of E229 in the micro receptor (in place of the corresponding D210 of the delta receptor) causes an adverse electrostatic interaction with C-terminal carboxylate-containing ligands, resulting in the observed preference of ligands with an uncharged C-terminus for the micro receptor. In addition, the requirement that the Phe3 side chain of JOM-13 assume a gauche orientation for optimal delta binding, whereas the Phe3 side chain of JOM-6 must be in a trans orientation for high-affinity micro binding can be largely attributed to the steric effect of replacement of L300 of the delta receptor by W318 of the micro receptor. Testing this hypothesis by examining the binding of JOM-6 and several of its key analogs with specific micro receptor mutants is described. Our initial results are consistent with the proposed ligand-receptor interaction models.

Differential effects of the novel non-peptidic opioid 4-tyrosylamido-6-benzyl-1,2,3,4 tetrahydroquinoline (CGPM-9) on in vitro rat t lymphocyte and macrophage functions.

Opioid receptors have been reported on immune cells of several species and shown to subserve effector functions of these cell types. Mu-selective opioid agonists such as morphine are immunosuppressive, whereas certain delta-opioid receptor-selective agonists have been associated with immunopotentiation. We have previously shown that intracerebroventricular administration of the non-peptidic delta-opioid receptor agonists did not alter certain parameters of immunocompetence. In this study, we evaluated the in vitro effects of the novel non-peptidic opioid 4-tyrosylamido-6-benzyl-1,2,3,4 tetrahydroquinoline (CGPM-9) on lymphocyte and macrophage functions. We demonstrated that CGPM-9 enhanced rat thymic lymphocyte proliferative response to concanavalin A (2.85- to 5.5-fold increases), and suppressed LPS-induced nitric oxide (67 to 72 percent reduction) and TNF-alpha production (46 percent reduction) by peritoneal macrophages, compared with untreated control. The mu-opioid receptor selective antagonist CTOP used at equimolar doses, significantly suppressed the effect of CGPM-9 on lymphocyte and macrophage functions (CTOP alone did not show any effect on lymphocyte or macrophage functions). In summary, CGPM-9 activated thymic lymphocyte proliferation and suppressed macrophage functions by acting at mu-opioid receptors. This suggests that opioid receptors on immunocytes may be coupled to different signaling pathways depending on the cell type and effector function being analyzed. The mechanism (s) associated with the differential effect of CGPM-9 on these immune cells remains to be elucidated. The pharmacotherapeutic potential for compounds such as CGPM-9 which potentiate T lymphocyte proliferation and suppress production of macrophage-derived inflammatory cytokines is substantial in research and clinical medicine.

Tetrapeptide derivatives of [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE) lacking an N-terminal tyrosine residue are agonists at the mu-opioid receptor.

The Phe(1) cyclic tetrapeptide Phe-c[D-Cys-Phe-D-Pen]NH(2) (Et) (JH-54) has been shown previously to exhibit high affinity and selectivity for the mu-opioid receptor. To examine the role of the Phe(1) residue in the unexpected high affinity of this peptide, 11 analogs of JH-54 have been synthesized and evaluated for opioid ligand binding and for efficacy using the [(35)S]GTPgammaS assay. Alteration of the bridging groups between the D-Cys(2) and D-Pen(4) residues of JH-54 from dithioether to disulfide revealed the importance of the relative position of the aromatic rings of the first and third residues in determining mu- and delta-affinities. The one carbon distance between the alpha carbon and phenyl ring in the N-terminal residue was critical. Additional steric bulk in the N-terminal Phe(1) residue was accommodated without large reductions in affinity in two naphthyl analogs, but not with 3, 3-(diphenyl)alanine. Conformational restriction of the Calpha-Cbeta and/or Cbeta-Cgamma bonds had little effect on affinities in two peptides with 2-amino-2-carboxytetralin in position 1, but it abolished activity in an isoquinoline analog and differentially altered activity in four phenylproline(1)-containing peptides. Most surprisingly, replacement of the Phe(1) aromatic ring with cyclohexyl resulted in a peptide of moderate affinity (K(i) = 32.5 nM) and potency (EC(50) = 58.8 nM). Thus, the tyrosyl para-hydroxyl substituent and even aromaticity in the N-terminal amino acid of these tetrapeptides are shown to be important, but not critical, features for mu-opioid receptor affinity, agonist potency, and efficacy.

Modifications of the cyclic mu receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]NH2 (Et): effects on opioid receptor binding and activation.

The previously described cyclic mu opioid receptor-selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]NH2 (Et) (JOM-6) was modified at residues 1 and 3 by substitution with various natural and synthetic amino acids, and/or by alteration of the cyclic system. Effects on mu and delta opioid receptor binding affinities, and on potencies and efficacies as measured by the [35S]-GTPgammaS assay, were evaluated. Affinities at mu and delta receptors were not influenced dramatically by substitution of Tyr1 with conformationally restricted phenolic amino acids. In the [35S]-GTPgammaS assay, all of the peptides tested exhibited a maximal response comparable with that of fentanyl at the mu opioid receptor, and all showed high potency, in the range 0.4-9nM. However, potency changes did not always correlate with affinity, suggesting that the conformation required for binding and the conformation required for activation of the opioid receptors are different. At the delta opioid receptor, none of the peptides were able to produce a response equivalent to that of the full delta agonist BW 373,U86 and only one had an EC50 value of less than 100nM. Lastly, we have identified a peptide, D-Hat-c[D-Cys-Phe-D-Pen]NH2 (Et), with high potency and > 1,000-fold functional selectivity for the mu over delta opioid receptor as measured by the [35S]-GTPgammaS assay.

Intracisternal nor-binaltorphimine distinguishes central and peripheral kappa-opioid antinociception in rhesus monkeys.

Systemic administration of nor-binaltorphimine (nor-BNI) produces a long-lasting kappa-opioid receptor (kappaOR) antagonism and has kappa(1)-selectivity in nonhuman primates. The aim of this study was to establish the pharmacological basis of central kappaOR antagonism in rhesus monkeys (Macaca mulatta). After intracisternal (i.c.) administration of small doses of nor-BNI, the duration and selectivity of nor-BNI antagonism were evaluated against two kappaOR agonists, (trans)-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488) and bremazocine. Thermal antinociception was measured in the warm water (50 degrees C) tail-withdrawal assay and sedation was evaluated by observers blind to treatment conditions. Following i.c. pretreatment with 0.32 mg nor-BNI, a 5- to 10-fold rightward shift of the U50,488 baseline dose-effect curve was observed in antinociception. In contrast, this dose of nor-BNI only produced an insignificant 2-fold shift against bremazocine. Pretreatment with a smaller dose (0.032 mg) of nor-BNI produced a 3-fold shift of U50, 488, which lasted for 7 days, but failed to alter the potency of bremazocine. This differential antagonism profile of i.c. nor-BNI also was observed in sedation ratings. In addition, the centrally effective dose of nor-BNI (0.32 mg), when administered s.c. in the back, did not antagonize either U50,488- or bremazocine-induced antinociception and sedation. After i.c. pretreatment with the same dose, nor-BNI also did not antagonize the peripherally mediated effect of U50,488 against capsaicin-induced thermal nociception in the tail. These results indicate that i.c. nor-BNI produces central kappaOR antagonism and support the notion of two functional kappaOR subtypes in the central nervous system. Moreover, it provides a valuable pharmacological basis for further characterizing different sources of kappaOR-mediated effects, namely, from central or peripheral nervous system receptors.

Prediction of protein structure: the problem of fold multiplicity.

Three-dimensional (3D) models of four CASP3 targets were calculated using a simple modeling procedure that includes prediction of regular secondary structure, analysis of possible beta-sheet topologies, assembly of amphiphilic helices and beta-sheets to bury their nonpolar surfaces, and adjustment of side-chain conformers and loops to provide close packing and saturation of the "hydrogen bond potential" (exposure of all polar groups to water or their involvement in intramolecular hydrogen bonds). It has been found that this approach allows construction of 3D models that, in some cases, properly reproduce the structural class of the protein (such as beta-barrel or beta-sandwich of definite shape and size) and details of tertiary structure (such as pairing of beta-strands), although all four models were more or less incorrect. Remarkably, some models had fewer water-exposed nonpolar side-chains, more hydrogen bonds, and smaller holes than the corresponding native structures (although the models had a larger water-accessible nonpolar surface). The results obtained indicate that hydrophobicity patterns do not unequivocally determine protein folds, and that any ab initio or fold recognition methods that operate with imprecise potential energy functions, or use crude geometrical approximations of the peptide chain, will probably produce many different nonnative structures.

The effects of the phyllolitorin analogue [desTrp(3), Leu(8)]phyllolitorin on scratching induced by bombesin and related peptides in rats.

Bombesin along with several closely related neuropeptides elicit scratching behavior when administered centrally. The first part of the study was designed to determine the antagonistic effects of a novel phyllolitorin analogue [desTrp(3),Leu(8)]phyllolitorin (DTP) on scratching induced by three peptides (bombesin, neuromedin-C, and [Leu(8)]phyllolitorin). In addition, the binding affinity of each peptide for the bombesin receptor site was determined. DTP (30 microg) inhibited scratching induced by these peptides, but unlike the peptides, DTP had no affinity for the bombesin site, thereby suggesting that DTP is displaying physiological antagonism through an unknown mechanism.

Structural organization of G-protein-coupled receptors.

Atomic-resolution structures of the transmembrane 7-alpha-helical domains of 26 G-protein-coupled receptors (GPCRs) (including opsins, cationic amine, melatonin, purine, chemokine, opioid, and glycoprotein hormone receptors and two related proteins, retinochrome and Duffy erythrocyte antigen) were calculated by distance geometry using interhelical hydrogen bonds formed by various proteins from the family and collectively applied as distance constraints, as described previously [Pogozheva et al., Biophys. J., 70 (1997) 1963]. The main structural features of the calculated GPCR models are described and illustrated by examples. Some of the features reflect physical interactions that are responsible for the structural stability of the transmembrane alpha-bundle: the formation of extensive networks of interhelical H-bonds and sulfur-aromatic clusters that are spatially organized as 'polarity gradients'; the close packing of side-chains throughout the transmembrane domain; and the formation of interhelical disulfide bonds in some receptors and a plausible Zn2+ binding center in retinochrome. Other features of the models are related to biological function and evolution of GPCRs: the formation of a common 'minicore' of 43 evolutionarily conserved residues; a multitude of correlated replacements throughout the transmembrane domain; an Na(+)-binding site in some receptors, and excellent complementarity of receptor binding pockets to many structurally dissimilar, conformationally constrained ligands, such as retinal, cyclic opioid peptides, and cationic amine ligands. The calculated models are in good agreement with numerous experimental data.

Complementarity of delta opioid ligand pharmacophore and receptor models.

The elaboration of a pharmacophore model for the delta opioid receptor selective ligand JOM-13 (Tyr-c[D-Cys-Phe-D-Pen]OH) and the parallel, independent development of a structural model of the delta receptor are summarized. Although the backbone conformation of JOM-13's tripeptide cycle is well defined, considerable conformational lability is evident in the Tyr(1) residue and in the Phe(3) side chain, key pharmacophore elements of the ligand. Replacement of these flexible features of the ligand by more conformationally restricted analogues and subsequent correlation of receptor binding and conformational properties allowed the number of possible binding conformations of JOM-13 to be reduced to two. Of these, one was chosen as more likely, based on its better superposition with other conformationally constrained delta receptor ligands. Our model of the delta opioid receptor, constructed using a general approach that we have developed for all rhodopsin-like G protein-coupled receptors, contains a large cavity within the transmembrane domain that displays excellent complementarity in both shape and polarity to JOM-13 and other delta ligands. This binding pocket, however, cannot accommodate the conformer of JOM-13 preferred from analysis of ligands, alone. Rather, only the "alternate" allowed conformer, identified from analysis of the ligands but "disfavored" because it does not permit simultaneous superposition of all pharmacophore elements of JOM-13 with other delta ligands, fits the binding site. These results argue against a simple view of a single, common fit to a receptor binding site and suggest, instead, that at least some binding site interactions of different ligands may differ.

Opioid receptor three-dimensional structures from distance geometry calculations with hydrogen bonding constraints.

Three-dimensional structures of the transmembrane, seven alpha-helical domains and extracellular loops of delta, mu, and kappa opioid receptors, were calculated using the distance geometry algorithm, with hydrogen bonding constraints based on the previously developed general model of the transmembrane alpha-bundle for rhodopsin-like G-protein coupled receptors (Biophys. J. 1997. 70:1963). Each calculated opioid receptor structure has an extensive network of interhelical hydrogen bonds and a ligand-binding crevice that is partially covered by a beta-hairpin formed by the second extracellular loop. The binding cavities consist of an inner "conserved region" composed of 18 residues that are identical in delta, mu, and kappa opioid receptors, and a peripheral "variable region," composed of 19 residues that are different in delta, mu, and kappa subtypes and are responsible for the subtype specificity of various ligands. Sixteen delta-, mu-, or kappa-selective, conformationally constrained peptide and nonpeptide opioid agonists and antagonists and affinity labels were fit into the binding pockets of the opioid receptors. All ligands considered have a similar spatial arrangement in the receptors, with the tyramine moiety of alkaloids or Tyr1 of opioid peptides interacting with conserved residues in the bottom of the pocket and the tyramine N+ and OH groups forming ionic interactions or H-bonds with a conserved aspartate from helix III and a conserved histidine from helix VI, respectively. The central, conformationally constrained fragments of the opioids (the disulfide-bridged cycles of the peptides and various ring structures in the nonpeptide ligands) are oriented approximately perpendicular to the tyramine and directed toward the extracellular surface. The results obtained are qualitatively consistent with ligand affinities, cross-linking studies, and mutagenesis data.

kappa-Opioid receptor binding populations in rhesus monkey brain: relationship to an assay of thermal antinociception.

The binding characteristics of the kappa opioid ligands [3H]U69,593 and [3H]bremazocine, the mu opioid ligand [3H][D-ala2,N-Me-Phe4,glycol5]enkephalin and the delta opioid ligand [3H]p-Cl-[D-pen2,5]enkephalin were studied in rhesus monkey brain membranes in saturation binding experiments and were followed by competition binding experiments with a variety of peptidic and nonpeptidic opioid ligands. The [3H]U69,593 sites appeared to be a subset of kappa opioid receptors (kappa-1 receptors: Kd, 1.2 nM; Bmax, 66 fmol/mg). [3H]Bremazocine (in the presence of mu and delta receptor-masking agents), bound to a larger population of kappa receptors (kappa-all: Kd, 0.39 nM; Bmax, 227 fmol/mg), which presumably included the aforementioned kappa-1 sites. Competition binding experiments revealed that the presently defined kappa-1 sites were similar to previously reported sites in other mammalian species, particularly in terms of the higher kappa-1 selectivity observed with arylacetamide (e.g., U50,488) vs. benzomorphan kappa agonists (e.g., ethylketocyclazocine). The kappa-selective antagonist norbinaltorphimine (nor-BNI) displayed a very small (2.3-fold) selectivity for kappa-1 vs. kappa-all sites. This led to the prediction that in rhesus monkeys (n = 3), systemically administered nor-BNI (10 mg/kg s.c.) should have a very moderate degree of antagonist selectivity for the antinociceptive effects of a putative kappa-1-agonist, the arylacetamide U50,488 (0.1-3.2 mg/kg s.c.), vs. those of the benzomorphan kappa agonist ethylketocyclazocine (0.01-056 mg/kg s.c.). This prediction was confirmed in vivo because nor-BNI (10 mg/kg) caused a robust and long lasting (up to 21 days) antagonism of the antinociceptive effects of U50,488 and a small but significant antagonism of ethylketocyclazocine. The arylacetamide congener Cl-977 (enadoline), which displayed an 11-fold kappa-1 vs. kappa-all binding selectivity, was not sensitive to nor-BNI pretreatment. This indicates that the kappa subtype-binding profile of an agonist is not necessarily predictive of its sensitivity to nor-BNI in vivo. Overall, the present results suggest that at least two functional kappa receptor populations may be present in rhesus monkey brain.

A high affinity, mu-opioid receptor-selective enkephalin analogue lacking an N-terminal tyrosine.

We report a high affinity, mu opioid receptor selective enkephalin analogue in which the N-terminal tyrosine residue thought to be required for such high affinity is replaced by phenylalanine. The high affinity can be traced to a shift of the ligand's N-terminal residue within the mu receptor binding pocket, which diminishes the importance of the usual hydrogen bond between the tyrosine phenolic moiety and the receptor.

Design of a high affinity peptidomimetic opioid agonist from peptide pharmacophore models.

A pair of diastereomeric peptidomimetics based upon opioid receptor-binding pharmacophore models derived for a series of opioid tetrapeptides was synthesized. Both analogues display high opioid receptor affinity, moderate selectivity for the mu opioid receptor, and are potent, full agonists.

Thermodynamic model of secondary structure for alpha-helical peptides and proteins.

A thermodynamic model describing formation of alpha-helices by peptides and proteins in the absence of specific tertiary interactions has been developed. The model combines free energy terms defining alpha-helix stability in aqueous solution and terms describing immersion of every helix or fragment of coil into a micelle or a nonpolar droplet created by the rest of protein to calculate averaged or lowest energy partitioning of the peptide chain into helical and coil fragments. The alpha-helix energy in water was calculated with parameters derived from peptide substitution and protein engineering data and using estimates of nonpolar contact areas between side chains. The energy of nonspecific hydrophobic interactions was estimated considering each alpha-helix or fragment of coil as freely floating in the spherical micelle or droplet, and using water/cyclohexane (for micelles) or adjustable (for proteins) side-chain transfer energies. The model was verified for 96 and 36 peptides studied by 1H-nmr spectroscopy in aqueous solution and in the presence of micelles, respectively ([set 1] and [set 2]) and for 30 mostly alpha-helical globular proteins ([set 3]). For peptides, the experimental helix locations were identified from the published medium-range nuclear Overhauser effects detected by 1H-nmr spectroscopy. For sets 1, 2, and 3, respectively, 93, 100, and 97% of helices were identified with average errors in calculation of helix boundaries of 1.3, 2.0, and 4.1 residues per helix and an average percentage of correctly calculated helix-coil states of 93, 89, and 81%, respectively. Analysis of adjustable parameters of the model (the entropy and enthalpy of the helix-coil transition, the transfer energy of the helix backbone, and parameters of the bound coil), determined by minimization of the average helix boundary deviation for each set of peptides or proteins, demonstrates that, unlike micelles, the interior of the effective protein droplet has solubility characteristics different from that for cyclohexane, does not bind fragments of coil, and lacks interfacial area.

The transmembrane 7-alpha-bundle of rhodopsin: distance geometry calculations with hydrogen bonding constraints.

A 3D model of the transmembrane 7-alpha-bundle of rhodopsin-like G-protein-coupled receptors (GPCRs) was calculated using an iterative distance geometry refinement with an evolving system of hydrogen bonds, formed by intramembrane polar side chains in various proteins of the family and collectively applied as distance constraints. The alpha-bundle structure thus obtained provides H bonding of nearly all buried polar side chains simultaneously in the 410 GPCRs considered. Forty evolutionarily conserved GPCR residues form a single continuous domain, with an aliphatic "core" surrounded by six clusters of polar and aromatic side chains. The 7-alpha-bundle of a specific GPCR can be calculated using its own set of H bonds as distance constraints and the common "average" model to restrain positions of the helices. The bovine rhodopsin model thus determined is closely packed, but has a few small polar cavities, presumably filled by water, and has a binding pocket that is complementary to 11-cis (6-s-cis, 12-s-trans, C = N anti)-retinal or to all-trans-retinal, depending on conformations of the Lys296 and Trp265 side chains. A suggested mechanism of rhodopsin photoactivation, triggered by the cis-trans isomerization of retinal, involves rotations of Glu134, Tyr223, Trp265, Lys296, and Tyr306 side chains and rearrangement of their H bonds. The model is in agreement with published electron cryomicroscopy, mutagenesis, chemical modification, cross-linking, Fourier transform infrared spectroscopy, Raman spectroscopy, electron paramagnetic resonance spectroscopy, NMR, and optical spectroscopy data. The rhodopsin model and the published structure of bacteriorhodopsin have very similar retinal-binding pockets.