Asp Side Chain Research Articles

Possible role for ligand binding of histidine 81 in the second transmembrane domain of the rat prostaglandin F 2α receptor

For the five principal prostanoids PGD 2, PGE 2, PGF 2α, prostacyclin and thromboxane A 2 eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF 2α receptor, which is conserved among all PGF 2α receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by site-directed mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF 2α with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF 2α. Wild type receptor and H81Q showed a shallow pH optimum for PGF2α binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF 2α with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF 2α receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor.

Electrostatic Effect on Electron Transfer at the Active Site of Heme Peroxidases: A Comparative Molecular Orbital Study on Cytochrome C Peroxidase and Ascorbate Peroxidase

Ab initio minimal basis set molecular orbital and discretized Poisson−Boltzmann electrostatic potential calculations were carried out on large models of the active site of heme peroxidases (cytochrome C peroxidase and cytosolic ascorbate peroxidase) to rationalize the location of the radical site in Compound I, an intermediate of the enzymatic reaction. Both molecular orbital and electrostatic potential calculations suggest that the spin distribution depends on the protonation state of the proximal His...Asp...Trp triad. If the transferable proton is shifted from the Trp side chain to Asp, the radical localizes on the indole group, while if it remains on the Trp the unpaired electron transfers to the heme group. Therefore, in cytochrome C peroxidase, Trp is deprotonated in Compound I, while it is protonated and neutral in cytosolic ascorbate peroxidase. Protonation state of the proximal residues is influenced by the electrostatic effect of the protein environment that differs in these enzymes, especially in the immediate vicinity of the Asp side chain. In contrast to earlier propositions we did not find evidence for the effect of the neighboring potassium-binding loop on the localization of the free radical. The heme peroxidases studied provide examples for the electrostatic, protonation-mediated modulation of electron transfer.

A model for the structure of the P domains in the subtilisin-like prohormone convertases.

The proprotein convertases are a family of at least seven calcium-dependent endoproteases that process a wide variety of precursor proteins in the secretory pathway. All members of this family possess an N-terminal proregion, a subtilisin-like catalytic module, and an additional downstream well-conserved region of approximately 150 amino acid residues, the P domain, which is not found in any other subtilase. The pro and catalytic domains cannot be expressed in the absence of the P domains; their thermodynamic instability may be attributable to the presence of large numbers of negatively charged Glu and Asp side chains in the substrate binding region for recognition of multibasic residue cleavage sites. Based on secondary structure predictions, we here propose that the P domains consist of 8-stranded beta-barrels with well-organized inner hydrophobic cores, and therefore are independently folded components of the proprotein convertases. We hypothesize further that the P domains are integrated through strong hydrophobic interactions with the catalytic domains, conferring structural stability and regulating the properties and activity of the convertases. A molecular model of these interdomain interactions is proposed in this report.

Aspartate 221 of thymidylate synthase is involved in folate cofactor binding and in catalysis.

Structural studies indicate that Asp 221 of Lactobacilluscasei thymidylate synthase forms a hydrogen bond network with the 2-amino and 3-imino groups of the folate [Matthews, D. A. (1990) J. Mol. Biol. 214, 937-948; Finer-Moore, J. S. (1990)Biochemistry 29, 6977-6986] that has been proposed to participate in catalysis. We prepared a complete replacement set of 19 mutants at position 221 of L. casei thymidylate synthase. Of these, the only one with sufficient activity to complement growth of a thymidylate synthase-deficient host was the Cys mutant. To further elucidate the function of the Asp 221 side chain, seven thymidylate synthase 221 mutants were studied in detail with regard to catalysis of dTMP formation and of thymidylate synthase partial reactions. Most of the mutants bound the nucleotide substrate dUMP with only moderate loss of binding affinity, indicating that the Asp side chain does not contribute to dUMP binding. Most of the mutants catalyzed the cofactor-independent dehalogenation of 5-bromodUMP; hence, the Asp side chain of TS is not essential for addition of the catalytic Cys residue to the nucleotide substrate. Mutants showed decreased affinity for the folate cofactor, but those with side chains capable of hydrogen bond formation were less severely affected. Some of the mutants were capable of forming covalent thymidylate synthase-5-fluorodUMP-methylenetetrahydrofolate complex; hence, the Asp side chain is not essential for steps leading to the covalent complex. We conclude that the hydrogen bond network between Asp 221 and the folate cofactor contributes to cofactor binding and a catalytic step after formation of the covalent ternary complex intermediate.

Involvement of glutamic acid 278 in the redox reaction of the cytochrome c oxidase from Paracoccus denitrificans investigated by FTIR spectroscopy.

The molecular processes concomitant with the redox reactions of wild-type and mutant cytochrome c oxidase from Paracoccus denitrificans were analyzed by a combination of protein electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy. Oxidized-minus-reduced FTIR difference spectra in the mid-infrared (4000-1000 cm-1) reflecting full or stepwise oxidation and reduction of the respective cofactor(s) were obtained. In the 1800-1000 cm-1 range, these FTIR difference spectra reflect changes of the polypeptide backbone geometry in the amide I (ca. 1620-1680 cm-1) and amide II (ca. 1560-1540 cm-1) region in response to the redox transition of the cofactor(s). In addition, several modes in the 1600-1200 cm-1 range can be tentatively attributed to heme modes. A peak at 1746 cm-1 associated with the oxidized form and a peak at 1734 cm-1 associated with the reduced form were previously discussed by us as proton transfer between Asp or Glu side chain modes in the course of the redox reaction of the enzyme [Hellwig, P., Rost, B., Kaiser, U., Ostermeier, C., Michel, H., and Mäntele, W. (1996) FEBS Lett. 385, 53-57]. These signals were resolved into several components associated with the oxidation of different cofactors. For a stepwise potential titration from the fully reduced state (-0.5 V) to the fully oxidized state (+0.5 V), a small component at 1738 cm-1 develops in the potential range of approximately +0.15 V and disappears at more positive potentials while the main component at 1746 cm-1 appears in the range of approximately +0.20 V (all potentials quoted vs Ag/AgCl/3 M KCl). This observation clearly indicates two different ionizable residues involved in redox-induced proton transfer. The major component at 1746 cm-1 is completely lost in the FTIR difference spectra of the Glu 278 Gln mutant enzyme. In the spectrum of the subunit I Glu 278 Asp mutant enzyme, the major component of the discussed difference band is lost. In contrast, the complete difference signal of the wild-type enzyme is preserved in the Asp 124 Asn, Asp 124 Ser, and Asp 399 Asn variants, which are critical residues in the discussed proton pump channel as suggested from structure and mutagenesis experiments. On the basis of these difference spectra of mutants, we present further evidence that glutamic acid 278 in subunit I is a crucial residue for the redox reaction. Potential titrations performed simultaneously for the IR and for the UV/VIS indicate that the signal related to Glu 278 is coupled to the electron transfer to/from heme a; however, additional involvement of CuB electron transfer cannot be excluded.

Protein–water–ion interactions in a model of the pore domain of a potassium channel: a simulation study

A model of the selectivity filter of a voltage-gated K + (Kv) channel formed by an eight-stranded β-barrel is compared with physiological properties of the channel. Continuum electrostatic calculations suggest that only two of the eight Asp sidechains at the extracellular mouth of the pore will ionise. A ring of four Tyr sidechains forms the narrowest region of the pore. Molecular dynamic simulations of the potential energy of a K + ion as translated along the model pore indicate that the two ionised Asp sidechains and the hydroxyl groups of the Tyr sidechains stabilise the partially desolvated ion as it passes through the narrowest region.

Specific amino acid recognition by aspartyl-tRNA synthetase studied by free energy simulations

Specific amino acid binding by aminoacyl-tRNA synthetases is necessary for correct translation of the genetic code. To obtain insight into the origin of the specificity, the binding to aspartyl-tRNA synthetase (AspRS) of the negatively charged substrate aspartic acid and the neutral analogue asparagine are compared by use of molecular dynamics and free energy simulations. Simulations of the Asn - AspRS complex show that although Asn cannot bind in the same position as Asp, several possible positions exist 1.5 to 2 Å away from the Asp site. The binding free energy of Asn in three of these positions was compared to that of Asp through alchemical free energy simulations, in which Asp is gradually mutated ito Asn in the complex with the enzyme. To correctly account for the electrostatic interactions in the system (including bulk solvent), a recently developed hybrid approach was used, in which the region of the mutation site is treated microscopically, whereas distant protein and solvent are treated by continuum electrostatics. Seven free energy simulations were performed in the protein and two in solution. The various Asn positions and orientations sampled at the Asn endpoints of the protein simulations yielded very similar free energy differences. The calculated Asp → Asn free energy change is 79.8(±1.5) kcal/mol in solution and 95.1(±2.8) kcal/mol in the complex with the protein. Thus, the substrate Asp is predicted to bind much more strongly than Asn, with a binding free energy difference of 15.3 kcal/mol. This implies that erroneous binding of Asn by AspRS is highly improbable, and cannot account for any errors in the translation of the genetic code. Almost all of the protein contributions to the Asp versus Asn binding free energy difference arise from an arginine and a lysine residue that hydrogen bond to the substrate carboxylate group and an Asp and a Glu that hydrogen bond to these; all four amino acid residues are completely conserved in AspRSs. The protein effectively “solvates” the Asp side-chain more strongly than water does. The simulations are analyzed to determine the interactions that Asn is able to make in the binding pocket, and which sequence differences between AspRS and the highly homologous AsnRS are important for modifying the amino acid specificity. A double or triple mutation of AspRS that could make it specific for Asn is proposed, and supported by preliminary simulations of a mutant complex.

Positional Preferences of Ionizable Residues in Gly-X-YTriplets of the Collagen Triple-helix

Collagens contain a high amount of charged residues involved in triple-helix stability, fibril formation, and ligand binding. The contribution of charged residues to stability was analyzed utilizing a host-guest peptide system with a single Gly-X-Y triplet embedded within Ac(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-Gly-NH2. The ionizable residues Arg, Lys, Glu, and Asp were incorporated into the X position of Gly-X-Hyp; in the Y position of Gly-Pro-Y; or as pairs of oppositely charged residues occupying X and Y positions. The Gly-X-Hyp peptides had similar thermal stabilities, only marginally less stable than Gly-Pro-Hyp, whereas Gly-Pro-Y peptides showed a wide thermal stability range (Tm = 30-45 degrees C). The stability of peptides with oppositely charged residues in the X and Y positions appears to reflect simple additivity of the individual residues, except when X is occupied by a basic residue and Y = Asp. The side chains of Glu, Lys, and Arg have the potential to form hydrogen bonds with available peptide backbone carbonyl groups within the triple-helix, whereas the shorter Asp side chain does not. This may relate to the unique involvement of Asp residues in energetically favorable ion pair formation. These studies clarify the dependence of triple-helix stability on the identity, position, and ionization state of charged residues.

Fourier-transform infrared spectroscopic studies on the coordination of the side-chain COO- groups to Ca2+ in equine lysozyme.

Interactions between Ca2+ and the Asp side chains in the Ca2+-binding site of equine lysozyme were investigated by Fourier-transform infrared (FT-IR) spectroscopy. In the spectrum of equine lysozyme, the intensities of the bands at about 1595 cm-1 and 1578 cm-1 in the region of the COO antisymmetric stretches increased upon Ca2+ binding. In the region of the COO- symmetric stretches, the loss of intensity at about 1388 cm-1 and gains of intensities at about 1423 cm-1 and 1403 cm-1 were observed due to Ca2+ binding to equine lysozyme. The spectral changes for equine lysozyme indicate that the COO- groups of Asp85, Asp90 and Asp91 in the Ca2+-binding site coordinate to Ca2+ in the pseudo-bridging mode, where divalent metal cation is bound to one of the two oxygens in the COO- group and a water molecule is hydrogen bonded to the other oxygen. The results presented here provide further evidence for a high degree of similarity between Ca2+-binding lysozyme and alpha-lactalbumin. The effects of Ca2+ binding on the main-chain conformation of equine lysozyme were compared with those of bovine alpha-lactalbumin and hen egg-white lysozyme.

Spirocyclic Peptidomimetics Featuring 2,3-Methanoamino Acids

The spirocyclic peptidomimetics cyclo-[{(2S,3S)-cyclo-R}GD{(2S,3S)-cyclo-R}GD] (3) and cyclo-[{(2R,3S)-cyclo-R}GD{(2R,3S)-cyclo-R}GD] (4), containing 2,3-methanoarginine (cyclo-Arg‘) residues, were prepared. Conformations of these molecules were studied via a combination of CD and 1D and 2D NMR, and these data were interfaced with extensive molecular simulations. Conformational biases for molecules 3 and 4 were thereby deduced and were compared with conclusions previously obtained (Burgess, K.; Lim, D.; Mousa, S. A. J. Med. Chem. 1996, 39, 4520) for cyclo-[RGDRGD] (2). Molecule 3 has a relatively clear conformational bias toward a structure with a type II β-turn between the Gly CO and Gly NH atoms, but 4 is more flexible, existing in conformers wherein the Asp and Arg side chains tend to be on opposite faces of the backbone cyclic structure. These conclusions were used to interpret the relative affinities of 2−4 with respect to the vitronectin receptor.

Molecular tuning of an EF-hand-like calcium binding loop. Contributions of the coordinating side chain at loop position 3.

Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca2+ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca2+ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca2+ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca2+ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning. 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics. Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity. Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain. Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.

A Computational Study of the Role of Solvation Effects in Reverse Turn Formation in the Tetrapeptides APGD and APGN

The tetrapeptides APGD and APGN are known by NMR analysis to adopt reverse turn conformations to a significant degree in aqueous solution. We have carried out a 7.7 ns molecular dynamics simulation of Ace-APGD-NHMe in explicit water, and have analyzed the energetics of snapshots from this simulation in terms of a molecular mechanics energy function, estimates of solvation free energy based on numerical solutions of the Poisson−Boltzmann equation (in which the solvent is treated as a high-dielectric continuum), and an estimate of chain entropy effects derived from a systematic search procedure. In the unconstrained trajectory, 17 transitions occur between turn and extended conformers, suggesting that the free energy profile is nearly flat and that the simulation is moderately-well-equilibrated with respect to this transition; the turn population found is within the experimental range. The potential of mean force, constructed as the sum of solute force-field energies, continuum solvation, and hard-sphere chain entropy, agrees with that computed directly from the simulation to within 2 kcal/mol across the entire range of configurations sampled. The study has been extended to the tetrapeptide APGN by repeating the energetic analysis with an Asn side chain replacing the Asp in the APGD snapshots. A comparison of energetics with Asp and Asn side chains shows a complex interplay among vacuum electrostatic terms, dielectric screening terms, and solvation free energy terms such that the net effect of side chain substitution on turn formation is very small. Prospects for application of this sort of analysis to other peptide and protein conformational problems are discussed.

The Negative Ion Mass Spectra of [M-H]− Ions Derived From Caeridin and Dynastin Peptides. Internal Backbone Cleavages Directed Through Asp and Asn Residues

The collision-induced spectra of [M–H]− ions of peptides containing Asp and Asn residues exhibit characteristic backbone cleavage ions produced via the enolate anions of the Asp or Asn side chains. Such reactions do not occur from the analogous Glu of Gln residues. Asp and Asn residues may be distinguished because the backbone cleavage ion formed from Asp undergoes pronounced loss of water: the corresponding loss of ammonia from the Asn backbone cleavage ion is less pronounced. The presence of Asp or Asn in a peptide usually results in the normal α and β backbone cleavage ions being minor in comparison to backbone cleavage ions formed via the enolate anions of Asp or Asn. © 1997 John Wiley & Sons, Ltd.

Structures of N-termini of helices in proteins.

We have surveyed 393 N-termini of alpha-helices and 156 N-termini of 3(10)-helices in 85 high resolution, non-homologous protein crystal structures for N-cap side-chain rotamer preferences, hydrogen bonding patterns, and solvent accessibilities. We find very strong rotamer preferences that are unique to N-cap sites. The following rules are generally observed for N-capping in alpha-helices: Thr and Ser N-cap side chains adopt the gauche - rotamer, hydrogen bond to the N3 NH and have psi restricted to 164 +/- 8 degrees. Asp and Asn N-cap side chains either adopt the gauche - rotamer and hydrogen bond to the N3 NH with psi = 172 +/- 10 degrees, or adopt the trans rotamer and hydrogen bond to both the N2 and N3 NH groups with psi = 1-7 +/- 19 degrees. With all other N-caps, the side chain is found in the gauche + rotamer so that the side chain does not interact unfavorably with the N-terminus by blocking solvation and psi is unrestricted. An i, i + 3 hydrogen bond from N3 NH to the N-cap backbone C = O in more likely to form at the N-terminus when an unfavorable N-cap is present. In the 3(10)-helix Asn and Asp remain favorable N-caps as they can hydrogen bond to the N2 NH while in the trans rotamer; in contrast, Ser and Thr are disfavored as their preferred hydrogen bonding partner (N3 NH) is inaccessible. This suggests that Ser is the optimum choice of N-cap when alpha-helix formation is to be encouraged while 3(10)-helix formation discouraged. The strong energetic and structural preferences found for N-caps, which differ greatly from positions within helix interiors, suggest that N-caps should be treated explicitly in any consideration of helical structure in peptides or proteins.

Ionization states of the catalytic residues in HIV-1 protease.

Chemical synthesis was used to prepare the HIV-1 protease specifically 13C-labelled in the catalytically essential Asp 25 in each monomer. The NMR chemical shift of the 13C-enriched homodimeric enzyme was measured in the presence of the inhibitor pepstatin, a mimic of the tetrahedral intermediate formed in enzyme catalysis. In this complex, the catalytic carboxyls do not titrate in the pH range where the enzyme is active; throughout the range pH 2.5-6.5, one Asp 25 side chain is protonated and the other deprotonated. By contrast, in the absence of inhibitor the two Asp side chains are chemically equivalent and both deprotonated at pH6, the optimum for enzymatic activity. These direct observations of the chemical properties of the catalytic apparatus of the enzyme provide concrete information on which to base the design of improved HIV-1 protease inhibitors.

Characterization of peptide-oligonucleotide heteroconjugates by mass spectrometry.

Two peptide-oligothymidylic acids, prepared by joining an 11 residue synthetic peptide containing one internal carboxyl group (Asp side chain) to amino-linker-5'pdT6 and amino-linker-5'pdT10 oligonucleotides, were analyzed by matrix-assisted laser desorption/ionization (MALDI) on a linear time-of-flight mass spectrometer and by electrospray ionization (ESI) on a triple-quadrupole system. These synthetic compounds model peptide-nucleic acid heteroconjugates encountered in antisense research and in studies that use photochemical crosslinking to investigate molecular aspects of protein-nucleic acid interactions. MALDI and ESI sensitivities for the two hybrid compounds were found to be similar respectively to their sensitivities for the pure oligonucleotide parts. In general, MALDI proved to be less affected by sample impurities and more sensitive than ESI, while ESI on the quadrupole produced greater mass accuracy and resolution than MALDI on the time-of-flight instrument. A hybrid's behavior in a MALDI-matrix or an ESI-spray-solvent was found to be governed mainly by the oligonucleotide. A single positive ESI tandem mass spectrum of the peptide-dT6 accounted for the heteroconjugate's entire primary structure including the point of the oligonucleotide's covalent attachment to the peptide.

Thermodynamic parameters of the reversible isomerization of aspartic residues via a succinimide derivative

Abstract Values of the thermodynamic quantities ΔH ° and ΔS ° for the reactions “Asp-peptide ⇆ Asupeptide ⇆ β-Asp-peptide” in aqueous solution have been obtained for the model peptides AcGlyXGlyGlyNHMe and AcXGlyNHMe (X  Asp, β-Asp, Asu; Asu = aminosuccinyl residue) from the temperature-dependence of equilibrium constants. The ΔH ° and ΔS ° values for the cyclization reactions of the carboxylic-acid form of the Asp and β-Asp side chains of the dipeptides and tetrapeptides are positive and coincident within experimental error. Medium values are 34 kJ mol −1 and 127 J K −1 mol −1 for ΔH ° and ΔS °, respectively. The molar enthalpies and molar entropies of the Asp-dipeptide and β-Asp-dipeptide, and of the Asp-tetrapeptide and β-Asp-tetrapeptide, did not exhibit significant differences.

Carboxyl group protonation upon reduction of the Paracoccus denitrificans cytochrome c oxidase: direct evidence by FTIR spectroscopy

The redox reactions of the cytochrome c oxidase from Paracoccus denitrificans were investigated in a thin-layer cell designed for the combination of electrochemistry under anaerobic conditions with UVIVIS and IR spectroscopy. Quantitative and reversible electrochemical reactions were obtained at a surface-modified electrode for all cofactors as indicated by the optical signals in the 400–700 nm range. Fourier transform infrared (FTIR) difference spectra of reduction and oxidation (reduced-minus-oxidized and oxidized-minus-reduced, respectively) obtained in the 1800-1000 cm −1 range reveal highly structured band features with major contributions in the amide I (1620–1680 cm −1) and amide II (1580-1520 cm −1) range which indicate structural rearrangements in the cofactor vicinity. However, the small amplitude of the IR difference signals indicates that these conformational changes are small and affect only individual peptide groups. In the spectral region above 1700 cm −1, a positive peak in the reduced state (1733 cm −1) and negative peak in the oxidized state (1745 cm −1) are characteristic for the formation and decay of a COOH mode upon reduction. The most obvious interpretation of this difference signal is proton uptake by one Asp or Gin side chain carboxyl group in the reduced state and deprotonation of another Asp or Glu residue. Moreover, both residues could well be coupled as a donor-acceptor pair in the proton transfer chain. An alternative interpretation is in terms of a protonated carboxyl group which shifts to a different environment in the reduced state. The relevance of this first direct observation of protein protonation changes in the cytochrome c oxidase for vectorial proton transfer and the catalytic reaction is discussed.

Conformational investigation of designed short linear peptides able to fold into β-hairpin structures in aqueous solution

Formation of secondary structure plays an important role in the early stages of protein folding. The conformational analysis of designed peptides has proved to be very useful for identifying the interactions responsible for the formation and stability of alpha-helices. However, very little is known about the factors leading to the formation of beta-hairpins. In order to get a good beta-hairpin-forming model peptide, two peptides were designed on the basis of beta-sheet propensities and individual statistical probabilities in the turn sites, together with solubility criteria. The conformational properties of the two peptides were analyzed by two-dimensional NMR methods. Long-range cross-correlations observed in NOE and ROE spectra, together with other NMR evidence, show that peptide IYSNPDGTWT forms a highly populated beta-hairpin in aqueous solution with a type I beta-turn plus a G1 beta-bulge conformation in the chain-bend region. The analogous peptide with a Pro5 substituted by Ser forms, in addition to the previous conformation, a second beta-hairpin with a standard type I beta-turn conformation, and the two forms are in fast dynamic equilibrium with one another. The effect of pH demonstrates the existence of a stabilizing interaction between the Asn and Asp sidechains. The populations of beta-hairpin conformations increase in the presence of trifluoroethanol (a structure-enhancing solvent). On the other hand, some residual structure persists at a high denaturant concentration (8 M urea). This work highlights the importance of the beta-turn residue composition in determining the particular type of beta-hairpin adopted by a peptide, though a role of interstrand sidechain interactions in the stabilization of the formed beta-hairpin is not discarded. The fact that trifluoroethanol can stabilize alpha-helices or beta-hairpins depending on the intrinsic properties of the peptide sequence is again shown. An additional example of the presence of residual structure under denaturing conditions is also presented.

Synthesis and solution conformation of cyclo[RGDRGD]: a cyclic peptide with selectivity for the alpha V beta 3 receptor.

Three peptides, cyclo[RGDRGD], cyclo[RGDRGd] (d = D-Asp), and the linear sequence RGDRGD, were prepared via solid phase syntheses. These were tested in binding assays based upon the alpha IIb/beta 3-fibrinogen and the alpha V beta 3-vitronectin interactions and found to be selective for the alpha V beta 3 integrin. The alpha V beta 3-vitronectin is important in bone regeneration, hence the compounds were also tested in an osteoclast regeneration assay; all three compounds, cyclo-[RGDRGD], cyclo[RGDRGd], and RGDRGD, showed modest activities. Molecular modeling, NMR, and CD studies were undertaken to elucidate the conformational preferences of cyclo-[RGDRGD], in aqueous solutions. Results from these studies strongly suggest that the molecule tends to adopt a type I beta-turn conformation with a relatively short distance between the Asp and Arg side chains. These observations are in harmony with the first correlations made between alpha V beta 3 selectivity and solution conformation for a peptide ligand (Pfaff, M.; et al. J. Biol. Chem. 1994, 269, 20233).