[Spectrum of pathogenic mtDNA mutations in Leber hereditary optic neuropathy families from Siberia].

The results of clinical, genealogical and molecular investigation of eighteen families with Leber hereditary optic neuropathy (LHON), identified on the territory of Siberia during the period from 1997 to 2005, are presented. Comprehensive analysis of mitochondrial genome variations in probands and their matrilineal relatives revealed the presence of relatively frequent (G11778A, G3460A, and T14484C), as well as rare and new mutations with the established or presumptive pathological effect (T10663C, G363A, C4640T, and A14619G). The G11778A mutation was detected in nine pedigrees (50%), mostly in the families of ethnic Russians. In eight of these families G11778A was found in preferred association with the coding-region substitutions, typical of western Eurasian mtDNA lineage (haplogroup) TJ. On the contrary, the G3460A mutation was detected in the three families belonging to the indigenous Siberian populations (Tuvinians, Altaians, and Buryats). It was associated with clearly different haplotypes of eastern Eurasian haplogroups, C3, D5, and D8. Unexpectedly, the G3460A de novo mutation was found in a large Tuvinian pedigree. At the same time, in eleven out of fourteen families of Caucasoid origin pathogenic mutations in the ND genes were associated with the T4216C and C1542A coding-region mutations, marking the root motif of haplogoup TJ. It is suggested that phylogenetically ancient mutations could have provided their carriers with the adaptive advantages upon the development of Central and Northern Europe at the end of the last glaciation (10 000 to 9 000 years ago), thereby, contributing to the preservation of weekly pathogenic LHON mutations, appearing at specific genetic background.

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.

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.

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.

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.

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.

pH changes in frog rods upon manipulation of putative pH-regulating transport mechanisms.

Rod intracellular pH (pHi) in the intact frog retina was measured fluorometrically with the dye 2',7'-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein under treatments chosen to affect putative pH-regulating transport mechanisms in the plasma membrane. The purpose was to relate possible pHi changes to previously reported effects on photoresponses. In nominally bicarbonate-free Ringer, application of amiloride (1 mM) or substitution of 95 mM external Na+ by K+ or choline triggered monotonic but reversible acidifications, consistent with inhibition of Na+/H+ exchange. Bicarbonate-dependent mechanisms were characterized as follows: (1) Replacing half of a 12 mM phosphate buffer by bicarbonate caused a sustained rise of pHi. (2) Subsequent application of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2',2'-disulphonic acid (DIDS, 0.2 mM) set off a slow acidification. (3) Substitution of external Cl- by gluconate (95 mM) caused a rapid pHi rise both in normal Na+ and low-Na+ perfusion. (4) This effect was inhibited by DIDS. The results support a consistent explanation of parallel electrophysiological experiments on the assumption that intracellular acidifications reduce and alkalinizations (in a certain range) augment photoresponses. It is concluded that both Na+/H+ exchange and bicarbonate transport control rod pHi, modulating the light-sensitive current. Part of the bicarbonate transport is by Na(+)-independent HCO3-/Cl- exchange, but a further Na(+)-coupled bicarbonate import mechanism is implicated.

Development of a model for the delta-opioid receptor pharmacophore. 4. Residue 3 dehydrophenylalanine analogues of Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13) confirm required gauche orientation of aromatic side chain.

We have previously proposed a model for the delta-opioid receptor binding conformation of the high affinity tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13) based on experimental and theoretical conformational analysis of this peptide and a correlation of conformational preferences of further conformationally restricted analogues of this tetrapeptide with their receptor binding affinities. A key element of this model is the requirement that the Phe3 side chain exist in the chi 1 = -60 degrees conformation. Conformational calculations on the residue 3 dehydrophenylalanine analogues of JOM-13 suggest that while the dehydro (Z) phenylalanine analogue can be superimposed easily with the proposed binding conformer of JOM-13, the dehydro(E)phenylalanine analogue cannot. These results lead to the prediction that the dehydro(Z)phenylalanine analogue should display similar delta-receptor binding affinity as JOM-13 while the dehydro(E)phenylalanine analogue is expected to bind less avidly. Synthesis and subsequent opioid receptor binding analysis of the dehydrophenylalanine analogues of JOM-13 confirm these predictions, lending support to the delta-pharmacophore model.

Development of a model for the delta-opioid receptor pharmacophore: 3. Comparison of the cyclic tetrapeptide, Tyr-c[D-Cys-Phe-D-Pen]OH with other conformationally constrained delta-receptor selective ligands.

We have previously proposed a model of the delta-opioid receptor bound conformation for the cyclic tetrapeptide, Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13) based on its conformational analysis and from conformation-affinity relationships observed for its analogues with modified first and third residues. To further verify the model, it is compared here with results of conformational and structure-activity studies for other known conformationally constrained delta-selective ligands: the cyclic pentapeptide agonist, Tyr-c[D-Pen-Gly-Phe-D-Phe]OH (DPDPE): the peptide antagonist, Tyr-Tic-Phe-PheOH (TIPP); the alkaloid agonist, 7-spiroindanyloxymorphone (SIOM); and the related alkaloid antagonist, oxymorphindole (OMI). A candidate delta-bound conformer is identified for DPDPE that provides spatial overlap of the functionally important N-terminal NH3+ and C-terminal COO- groups and the aromatic rings of the Tyr and Phe residues in both cyclic peptides. It is shown that all delta-selective ligands considered have similar arrangements of their pharmacophoric elements, i.e., the tyramine moiety and a second aromatic ring (i.e., the rings of Phe3, Phe4, and Tic2 residues in JOM-13, DPDPE, and TIPP, respectively; the indole ring system in OMI, and the indanyl ring system in SIOM). The second aromatic rings, while occupying similar regions of space throughout the analogues considered, have different orientations in agonists and antagonists, but identical orientations in peptide and alkaloid ligands with the same agonistic or antagonistic properties. These results agree with the previously proposed binding model for JOM-13, are consistent with the view that delta-opioid agonists and antagonists share the same binding site, and support the hypothesis of a similar mode of binding for opioid peptides and alkaloids.

Sulfhydryl binding reagents increase the conductivity of the light-sensitive channel and inhibit phototransduction in retinal rods.

The mechanisms by which sulfhydryl (SH-) binding reagents modulate the light-sensitive conductance of retinal rods were investigated by current recording from single rods, by patch clamp recording from the plasma membrane of the rod outer segment (ROS), and by biochemical study of their effects on the light-induced hydrolysis of cyclic GMP. The electrophysiology, as well as measurements of the reagents' ability to traverse the ROS plasma membrane, was done on amphibian (Rana and Ambystoma) rods, and the biochemistry on bovine rods. The main SH-reagents used were N-ethyl-maleimide (NEM) and iodoacetamide (IAA). Both transiently increased rod current, but part of the large current could not be turned off by light. After a few minutes' exposure, NEM, but not IAA, caused a continuous decay of the rod's light sensitivity. In patch-clamp recordings from the ROS plasma membrane, the reagents increased conductivity both in the presence and absence of cGMP, consistent with the observation that the drug-induced current increase in intact rods involved both light-sensitive and light-insensitive components. In vitro, NEM was found to be a powerful inhibitor of cGMP hydrolysis, which can explain the gradual loss of light sensitivity in the rod and could initially contribute to the increased dark current via elevated cGMP levels. Thus, SH-reagents act both by modifying the light-sensitive channel and by inhibiting phototransduction inside the rod.

[Aggregation of rhodopsin molecules during damaging exposure of photoreceptor membranes to light]. / Agregatsiia molekul rodopsina pri povrezhdaiushchem deistvii sveta na fotoretseptornye membrany.

Injuring light induced structural changes in rod outer segment (ROS) membranes are studied using "ST EST spectroscopy" for spin labelled rhodopsin, ESR of lipid spin label and SDS gel-electrophoresis. Free SH-group content of rhodopsin and lipid peroxidation level were simultaneously determined as well. A decrease of rotational mobility of rhodopsin in ROS induced by prolonged illumination is shown to result from irreversible protein aggregation caused by disulfide bond formation between "hydrophobic" SH-groups of rhodopsin. Some decrease of lipid microviscosity and degree of order are found, in contrast to considerable rise in microviscosity due to Fe2+-ascorbate induced lipid peroxidation of ROS membranes. Lipid oxidation is found to accelerate protein aggregation which in its turn influences the state of lipid bilayer.

[Photodamage of rhodopsin molecule. Oxidation of SH-group]. / Fotopovrezhdenie molekuly rodopsina. Okislenie SH-grupp.

Illumination of rod outer segments with bright visible light results in the oxidation of both protein and lipid components of the photoreceptor membrane. The oxidation degree depends on the intensity and time of illumination. The inhibitors of free radical processes completely inhibit lipid oxidation and somewhat decrease protein oxidation. Photooxidation systems of lipids and rhodopsin also react differently to oxygen content in the incubation medium. Retinal is the photosensitizer of the oxidation of the photoreceptor membrane components.