Conformation of peptides constructed from achiral amino acid residues Aib and DeltaZPhe: computational study of the effect of L/D- Leu at terminal positions

Conformational studies of the peptides constructed from achiral amino acid residues Aib and Delta(Z)Phe (I) Ac-Aib-Delta(Z)Phe-NHMe (II), and Ac-(Aib-Delta(Z)Phe)(3)-NHMe; peptides III-VI having L-Leu or D-Leu at either the N- or the C-terminal position and of peptides VII-X having Leu residues in different enantiomeric combinations at both the N- and the C-terminal positions in peptide II have been studied to design the peptide with the required helical sense. Peptide II, as expected, adopts degenerate left- and right-handed helical structures. It has been shown that the peptides IV and VI having D-Leu at either the N or the C terminus can be realized in the right-handed helical structure with the phi,psi values of -20 degrees and -60 degrees for the Aib/Delta(Z)Phe residues. L-Leu and D- Leu at both the terminals in peptides VII and VIII, respectively, have hardly any effect as both the left- and the right-handed structures are found to be degenerate. Peptides III and IX can be realized in right- and left-handed helical structures, respectively, in solvents of low polarity whereas peptides V and X are predicted to be in the right-handed helical structures stabilized by carbonyl-carbonyl interactions without the formation of hydrogen bonds. The conformational states with the phi,psi values of 0 degrees and -85 degrees in peptide V are characterized by rise per residue of 2.03 A, rotation per residue of 117.5 degrees , and 3.06 residues per turn. In all peptides having Leu residue at the N terminus, the methyl moiety of the acetyl group is involved in the CH/pi interactions with the Cepsilon--Cdelta edge of the aromatic ring of Delta(Z)Phe (3) and the amino group NH of Delta(Z)Phe is involved in the NH/pi interactions with its own aromatic ring. The CH(3) groups of the Aib residues are also involved in CH/pi interactions with the i + 1th and i + 3th Delta(Z)Phe's aromatic side chains.     

Synthesis, crystal structure and molecular conformation of Nalpha-Boc-L-leucyl-(Z)-beta-(3-pyridyl)-alpha,beta- dehydroalanyl-L-leucine methyl ester.

A protected tridehydropeptide containing (Z)-beta-(3-pyridyl)-alpha,beta-dehydroalanine (Delta(Z)3Pal) residue, Boc-Leu-Delta(Z)3Pal-Leu-OMe (1), was synthesized via Erlenmeyer azlactone method. X-ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Delta(Z)Phe analog, Boc-Leu-Delta(Z)Phe-Leu-OMe (2).     

Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions

The structural origins of the specificity of the neurophysin hormone-binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide alpha-amino group, two factors are considered central. (1) The bound Leu side chain can establish only one-third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition.     

Structural and conformational properties of (Z)-beta-(1-naphthyl)- dehydroalanine residue

To understand how chemical structure of beta-substituted alpha, beta-dehydroalanine (particularly size and pi conjugation of beta substituent) affects conformational property, x-ray crystallographic analysis was performed on Boc-Ala-Delta(Z) Nap-Val-OMe [Boc: t-butoxycarbonyl; Delta(Z) Nap: (Z)-beta-(1-naphthyl)dehydroalanine; OMe: methoxy] having the naphthyl group as a bulky beta substituent. Single crystals were grown by slow evaporation from an ethanol solution in the triclinic space group P1 with a = 9.528 (3) A, b = 12.410(4) A, c = 5.975(2) A, alpha = 96.77(3) degrees, beta = 102. 81(2) degrees, gamma = 88.74(3) degrees, V = 684.1(4) A3, and Z = 1. Phase determination was carried out by a direct method (SHELEXS), and the final structure was refined to R = 8.1% and R(w) = 9.0% for 1964 observed reflections. The bond lengths and bond angles of the Delta(Z)Nap residue, characterized by a sp(2) hybridized C(alpha) atom, did not differ from those of other dehydroresidues such as Delta(Z) Phe, Delta(Z) Leu, and DeltaVal essentially. The peptide backbone took a type II beta-turn conformation involving an intramolecular hydrogen bond between CO(Boc) and NH(Val), similar to di- or tripeptides containing a Delta(Z) Phe or Delta(Z) Leu residue in the second positions. Here the naphthyl group was found to be nonplanar [chi(2) = 55(1) degrees ] relative to the C(alpha)==C(beta)==C(gamma) plane. The nonplanarity was supported by conformational energy calculation. The molecular packing was stabilized by two kinds of intermolecular hydrogen bonds and van der Waals interactions. Naphthyl groups were arranged in a partially overlapped face-to-face orientation with a center-to-center distance of 5.97 A. For additional information, peptide Boc-(Ala-Delta(Z) Nap-Leu)(2)-OMe was synthesized and its solution conformation was investigated by (1)H-NMR spectroscopy. The hexapeptide showed the tendency to form a 3(10)-helical conformation in solution essentially. Conformational properties of Delta(Z) Nap residue, characterized by a type II beta-turn and 3(10)-helix, were supported by a conformational energy contour map of the Delta(Z)Nap residue.     

External chirality-triggered helicity control promoted by introducing a beta-Ala residue into the N-terminus of chiral peptides

The noncovalent chiral domino effect (NCDE), defined as chiral interaction upon an N-terminus of a 3(10)-helical peptide, will provide a unique method for structural control of a peptide helix through the use of external chirality. On the other hand, the NCDE has not been considered to be effective for the helicity control of peptides strongly favoring a one-handed screw sense. We here aim to promote the NCDE on peptide helicity using two types of nonapeptides: H-beta-Ala-Delta(Z)Phe-Aib-Delta(Z)Phe-X-(Delta(Z)Phe-Aib)(2)-OCH(3) [Delta(Z)Phe = alpha,beta-didehydrophenylalanine, Aib = alpha-aminoisobutyric acid], where X as the single chirality is L-leucine (1) or L-phenylalanine (2). NMR, IR, and CD spectroscopy as well as energy calculation revealed that both peptides alone form a right-handed 3(10)-helix. The original CD amplitudes or signs in chloroform, irrespective of a strong screw-sense preference in the central chirality, responded sensitively to external chiral information. Namely added Boc-L-amino acid stabilized the original right-handed helix, while the corresponding d-isomer destabilized it or transformed it into a left-handed helix. These peptides were also shown to bind more favorably to an L-isomer from the racemate. Although similar helicity control was observed for analogous nonapeptides bearing an N-terminal Aib residue (Inai, Y.; et al. Biomacromolecules 2003, 4, 122), the present findings demonstrate that the N-terminal replacement by the beta-Ala residue significantly improves the previous NCDE to achieve more effective control of helicity. Semiempirical molecular orbital calculations on complexation of peptide 2 with Boc-(L or D)-Pro-OH reasonably explained the unique conformational change induced by external chirality.     

Changes at the floor of the peptide-binding groove induce a strong preference for proline at position 3 of the bound peptide: molecular dynamics simulations of HLA-A*0217

We report on molecular dynamics simulations of major histocompatibility complex (MHC)-peptide complexes. Class I MHC molecules play an important role in cellular immunity by presenting antigenic peptides to cytotoxic T cells. Pockets in the peptide-binding groove of MHC molecules accommodate anchor side chains of the bound peptide. Amino acid substitutions in MHC affect differences in the peptide-anchor motifs. HLA-A*0217, human MHC class I molecule, differs from HLA-A*0201 only by three amino acid residues substitutions (positions 95, 97, and 99) at the floor of the peptide-binding groove. A*0217 showed a strong preference for Pro at position 3 (p3) and accepted Phe at p9 of its peptide ligands, but these preferences have not been found in other HLA-A2 ligands. To reveal the structural mechanism of these observations, the A*0217-peptide complexes were simulated by 1000 ps molecular dynamics at 300 K with explicit solvent molecules and compared with those of the A*0201-peptide complexes. We examined the distances between the anchor side chain of the bound peptide and the pocket, and the rms fluctuations of the bound peptides and the HLA molecules. On the basis of the results from our simulations, we propose that Pro at p3 serves as an optimum residue to lock the dominant anchor residue (p9) tightly into pocket F and to hold the peptide in the binding groove, rather than a secondary anchor residue fitting optimally the complementary pocket. We also found that Phe at p9 is used to occupy the space created by replacements of three amino acid residues at the floor within the groove. These findings would provide a novel understanding in the peptide-binding motifs of class I MHC molecules.     

A characteristic property of vitamin K-dependent plasma protein Z

Evidence is presented for rapid, limited proteolysis of protein Z by alpha-thrombin. This alpha-thrombin-catalyzed proteolysis of protein Z occurred at a single peptide linkage, between Arg-365 and Gly-366, located in the COOH-terminal portion. The resulting NH2-terminal large fragment (PZt) and the COOH-terminal peptide (C-peptide) were isolated and chemically characterized. The C-peptide consisted of 31 amino acid residues including one galactosamine-type Thr residue and was assigned to the position from Gly-366 to the COOH-terminal residue of Val-396 in protein Z. The NH2-terminal large fragment, PZt, constituted the remainder of protein Z. The abilities to bind calcium of intact protein Z, PZt, and the derivative of protein Z devoid of the NH2-terminal gamma-carboxyglutamic acid (Gla) domain (Gla-domainless), prepared with the known chymotrypsin treatment, were examined by equilibrium dialysis. The results indicated that intact protein Z and PZt contain four calcium binding sites with dissociation constants of 0.1 mM. Moreover, the Scatchard plot analysis showed positive cooperativity, suggesting the presence of at least two initial sites for calcium binding. In contrast, the Gla-domainless protein Z had no calcium binding site, indicating that the domain of protein Z functional for calcium binding occurs within the NH2-terminal Gla domain. This differed from factor X, factor IX, protein S, and protein C, all of which contain one or two calcium binding site(s) independent on their Gla-domains.     

Effect of terminal achiral and chiral residues on the conformational behaviour of poly Δ(z)Phe and analysis of various interactions

Conformational properties of the peptides containing (Δ(Z)Phe)6 with achiral (ΔAla, Gly) and chiral (Ala, Leu) residues at both the N- and C-terminal positions have been studied with a view to design a peptide with desired helical screw sense. In all the peptides, the lowest energy conformational state corresponds to Φ = 0° and Ψ = + 90° or - 90° or both +/- 90°. These structures are characterized by rise per residue of 1.94 ?; rotation per residue of 114° and 3.12 residues per turn and are stabilized by: (i) carbonyl-carbonyl interactions with the carbonyl oxygen of ith residue and carbonyl carbon atom of the carbonyl group of ith+1 residue; and (ii) N-H....π interactions between the amino group of Δ(Z)Phe and its own aromatic moiety. The Ala/Leu residues at the N-terminus further stabilized the structure, through C-H....π interactions with the farthest edge of the aromatic ring of ith+3 Δ(Z)Phe residue. For peptides Ac-L-Ala/L-Leu-(Δ(Z)Phe)6-NHMe, the low energy left handed helical structure (approximately 2.5 Kcalmol?1 higher in energy) state corresponds to Φ = -30°, Ψ = 120° for L-residue and Φ = Ψ = 30° for Δ(Z)Phe residues and is in good agreement with the X-ray crystallography results for the peptide Boc-L-Ala-(Δ(Z)Phe)4-NHMe crystals grown from acetonitrile/ethanol mixture. Computational results suggest that the peptides Ac-DAla/D-Leu-(Δ(Z)Phe)6-NHMe adopt a right handed helical structure in polar solvents with Φ = 30°, Ψ = -120° for D-residues and Φ = Ψ = -30° for Δ(Z)Phe residues. Both in the left handed and right handed structures, the carbonyl oxygen of acetyl group is involved in 10-membered hydrogen bonded ring formation with NH of 3rd Δ(z)Phe residue whereas Δ(Z)Phe residues backbone adopts a 3?? helix structure. Computational results also suggest that the conformational state with Φ = 0° and Ψ = 90° can be realized by keeping D-Ala or D-Leu at the C-terminal. There is hardly any effect of achiral residues Gly/ΔAla on the conformational behaviour of poly-Δ(Z)Phe.     

Effect of Stereochemistry (Z and E) and Position of α,β-Dehydrophenylalanine (ΔPhe) on β-turn Stability

Several model peptides containing α, β-dehydrophenylalanine (ΔPhe) in both Z and E configurations were studied for β-turn stability at the AM1 level of theory. Both configurations of ΔPhe are well able to stabilize β-turns in the backbone. However, the β-turns for peptides bearing Z-ΔPhe are energetically more stable than the E-counterparts. The difference in energies between the global minima of these peptides having the Z and E configuration of ΔPhe, is dictated by the size and stereochemistry of residues flanking ΔPhe. One distinct feature of E-ΔPhe is that it pushes peptides to adopt a Type II β-turn with the ΔPhe residue in the (i + 1) position of the turn. This unique feature may be exploited in peptide design.     

Characterization of three pheromone-binding proteins (PBPs) of Helicoverpa armigera (Hübner) and their binding properties

Three pheromone-binding proteins of Helicoverpa armigera were cloned and expressed in Escherichia coli. In order to characterize their physiological properties, ligand-binding experiments were performed using five biologically relevant substances including sex pheromones and interspecific signals. The results showed that one of the pheromone-binding proteins, HarmPBP1, binds strongly to each of the two principal pheromone components of H. armigera, (Z)-11-tetradecenal and (Z)-9-hexadecenal, but not to the interspecific signal (Z)-9-tetracecenal. The two remaining pheromone-binding proteins, HarmPBP2 and HarmPBP3, showed only weak affinities with the ligands tested. The 3-D structure of HarmPBP1 was predicted and the docking experiments indicate that the key binding site of (Z)-9-hexadecenal to HarmPBP1 includes Thr112, Lys111, and Phe119 whereas that of (Z)-11-tetradecenal includes Ser9, Trp37, Phe36, and Phe119.     

Structure-activity relationships of Leu-Enkephalin analog with (4-Carboxamido)phenylalanine substituted for tyrosine: a molecular dynamics study

Motivated by recent experimental work on Leu-Enkephalin modification with (4-Carboxamido)phenylalanine (Cpa), we perform MD simulations to study the structure-activity relationships of the [Cpa(1), Leu(5)]-enkephalin (Cpa-LE) for better understandings of the binding affinity in delta-selective opioid ligands. Recently, Tyr(1) substituted into Cpa(1) form was experimentally found to be the first example of an amino acid that acts as a surrogate for Tyr(1) in opioid peptide ligands, which challenges a long-standing belief that a phenolic residue is required for high affinity binding. Our simulations show the Cpa-LE structure in aqueous solution revealed that the occurrence of single-bend packed state can be stabilized by an intramolecular hydrogen bond from Leu(5)-NH to Gly(2)-CO (5-->2). In addition, an intramolecular sidechain to backbone hydrogen bond, i.e., hydrogen bond binding between the sidechain carbonyl CO group of the Cpa residue and backbone amide NH group of the Phe residue was examined. Furthermore, the hydration effects of carboxamido group (CONH(2)) for Cpa residue and 5-->2 hydrogen bond were calculated via the solute-solvent radial distribution functions g(alpha-beta) (r), providing direct evidence of strong hydrogen bond interactions. Our simulation results further reveal the chi(1) rotamers of the Cpa(1) and Phe(4) that show preferences for trans and gauche (-), respectively. Finally, we elucidate the probability distributions of two aromatic rings among the Cpa-LE, Leu-enkephalin, and delta pharmacophore model. The results show that modified the Tyr(1) to Cpa(1) can lead to increase the potency and selectivity for delta-opioid receptor (DOR), consistent with experimental findings.     

NMR studies of 4-19F-phenylalanine-labeled intestinal fatty acid binding protein: evidence for conformational heterogeneity in the native state

(19)F-Nuclear magnetic resonance (NMR) studies have been carried out after incorporation of 4-(19)F-phenylalanine into the intestinal fatty acid binding protein (IFABP), a protein composed of two beta-sheets containing a large hydrophobic cavity into which ligands bind. NMR spectra have been obtained with both the ligand-free and ligand-bound (oleate) forms. There are 29 residues involved in van der Waals or hydrophobic interactions or both to form a U-shaped ligand binding pocket (Sacchettni J. C., Scapin G., Gopaul D., and Gordon J. I. (1992) J. Biol. Chem. 267, 23534-23545). The protein contains eight phenylalanines, and all are included in those residues that line the pocket. Peak assignments were made using site-specific incorporation of 4-(19)F-phenylalanine. Fluorine is a highly sensitive probe to monitor the conformation and dynamics of the side chains in native state. We find that chemical exchange in the binding pocket exists in the native apo- and holo-state. Of the eight phenylalanine residues, Phe2, Phe47, Phe62, Phe68, and Phe93 are arranged on one side of the binding pocket, and all exist in two conformations with Phe2, Phe47, and Phe62 showing exchange cross-peaks with minor conformation in (19)F-(19)F nuclear Overhauser effect (NOESY) spectra. The line widths of Phe68 and Phe93 are broader than those of other phenylalanine residues and can be deconvoluted into two peaks. Phe47, Phe62, Phe68, Phe93, and Trp82 have been proposed to be involved in the early stage of collapse (Ropson, I. J., and Frieden, C. (1992) Proc. Natl. Acad. Sci U.S.A. 89, 7222-7226), but a temperature study suggests that Phe47 behaves differently than other residues and may be more involved in a later stage of folding, for example, side chain stabilization. In the holo-form, Phe17 shows an extra exchange cross-peak in addition to those exchange cross-peaks observed in apo-form. Holo-IFABP exhibits broader line width than the apo-form, suggesting more flexibility of the binding cavity upon ligand binding.     

Synthesis and binding properties of specific photoaffinity ligands for mu and delta opioid receptor subtypes

We report the synthesis and binding properties of specific photoaffinity ligands for mu and delta opioid receptor subtypes. These ligands are derived from DAGO: Tyr-D-Ala-Gly-NMePhe-Gly-ol, a mu selective probe and DTLET: Tyr-D-Thr-Gly-Phe-Leu-Thr, a delta selective probe by modifying the Phe 4 residue. These modifications are: i) a nitro group on the para position of Phe ring as Phe(4 NO2) or Nip, ii) an azido group as Phe(4 N3) or AZ. Pharmacological responses on mouse vas deferens (delta sites) and guinea pig ileum (mu sites), as well as competition experiments with [3H] DAGO and [3H] DTLET on crude rat brain membranes have been performed. The nitro group on the phenyl ring of the Phe residue preserves the affinity and selectivity of each probe: NipDAGO for the mu sites, NipDTLET for the delta ones. However the nitro probes do not appear to be photoactivable by u.v. irradiation. Likewise, azidation of the phenyl ring of the Phe residue does not change the receptor selectivity of each probe, but AZDAGO has less affinity than its parent molecule DAGO, while AZDTLET has more affinity than DTLET. These compounds are photoactivable and provide an efficient tool to characterize and isolate the different receptor subtypes, especially the delta site.     

Complex of an active mu-opioid receptor with a cyclic peptide agonist modeled from experimental constraints

Site-directed mutagenesis and design of Zn(2+)-binding centers have been used to determine a set of specific tertiary interactions between the mu-opioid receptor, a rhodopsin-like G protein-coupled receptor (GPCR), and its cyclic peptide agonist ligand, Tyr(1)-c(S-Et-S)[d-Cys(2)-Phe(3)-d-Pen(4)]NH(2) (JOM6). The binding affinity of the tetrapeptide is strongly dependent on the nature of its first and third residues and on substitutions at positions 213, 216, 237, 300, 315, and 318 of the mu-opioid receptor. His(1) and His(3) analogues of the ligand were able to form metal-binding complexes with the V300C and G213C/T315C receptor mutants, respectively. Direct contact of the Phe(3) residue of JOM6 with Gly(213), Asp(216), Thr(315), and Trp(318) of the receptor was suggested by the binding affinities of His(3)-, Nle(3)-, Leu(3)-, Aci(3)-, Delta(E)Phe(3)-, and Delta(Z)Phe(3)-substituted peptides with the G213C/T315C, D216V, T315C, and W318L mutants. The improved binding affinity of the free carboxylate analogue of JOM6 for binding to the E229D mutant revealed an interaction between the C-terminal group of the peptide and Glu(229) of the receptor. The experimental constraints that were obtained were applied for distance geometry modeling of the mu-receptor in complex with the tetrapeptide agonist ligand, JOM6. The active conformation of the opioid receptor was calculated using the crystal structure of "inactive" rhodopsin and published engineered and intrinsic metal-binding sites and disulfide bonds that allow or facilitate activation of GPCRs. Interhelical H-bonds existing in the mu-receptor were applied as additional distance constraints. The calculated model of the receptor-ligand complex can serve as a prototype of the active state for all rhodopsin-like GPCRs. It displays a strongly shifted transmembrane helix 6 (TM6) and reorientation of the conserved Trp(293) residue in TM6 upon its interaction with the agonist. Importantly, the binding pockets of the active and inactive states are not identical, which implies distinct interaction modes of agonists and antagonists. In the active state, the binding pocket of the mu-receptor is complementary to the previously proposed receptor-bound conformation of JOM6.     

Chiral interaction in Gly-capped N-terminal motif of 3(10)-helix and domino-type induction in helix sense

Chiral interaction of helical peptide with chiral molecule, and concomitant induction in its helix sense have been demonstrated in optically inactive nonapeptide (1) possessing Gly at its N-terminus: H-Gly-(Delta(Z)Phe-Aib)(4)-OCH(3) (1: Delta(Z)Phe = Z-dehydrophenylalanine; Aib = alpha-aminoisobutyric acid). Spectroscopic measurements [mainly nuclear magnetic resonance (NMR) and circular diochroism (CD)] as well as theoretical simulation have been carried out for that purpose. Peptide 1 in the 3(10)-helix tends to adopt preferentially a right-handed screw sense by chiral Boc-L-amino acid (Boc: t-butoxycarbonyl). Induction in the helix sense through the noncovalent chiral domino effect should be derived primarily from the complex supported by the three-point coordination on the N-terminal sequence. Thus the 3(10)-helical terminus consisting of only alpha-amino acid residues enables chiral recognition of the Boc-amino acid molecule, leading to modulation of the original chain asymmetry. Dynamics in the helix-sense induction also have been discussed on the basis of a low-temperature NMR study. Furthermore, the inversion of induced helix sense has been achieved through solvent effects.     

Combined effect of the DeltaPhe or DeltaAla residue and the p-nitroanilide group on a didehydropeptides conformation

Two series of dehydropeptides of the general formulae Boc-Gly-X-Phe-p-NA, Boc-Gly-Gly-X-Phe-p-NA, Gly-X-Gly-Phe-p-NA.TFA, and Boc-Gly-X-Gly-Phe-p-NA, with X = Delta(Z)Phe and DeltaAla, were studied with NMR in DMSO and CDCl(3)-DMSO, and with CD in MeOH, MeCN, and TFE. The NMR spectra measured in DMSO suggest that peptides with the DeltaPhe residue next to Phe are folded whereas peptides with Gly between DeltaPhe and Phe are less ordered. NMR spectra of DeltaAla-containing peptides indicate that these peptides are flexible and their conformational equilibria are populated by many different conformations. The CD spectra show that conformational properties of the peptides studied are distinctly influenced by a mutual position of the dehydroamino acid residue and the p-NA group. They indicate that all dehydropeptides with the DeltaPhe residue, Boc-Gly-DeltaAla-Phe-p-NA, and Boc-Gly-Gly-DeltaAla-Phe-p-NA adopt ordered conformations in all solvents studied, presumably of the beta-turn type. The last two peptides exhibit surprising chiroptical properties. Their spectra show exciton coupling-like couplets in the region of the p-NA group absorption. This shape of CD spectra suggests a rigid, chiral conformation with a fixed disposition of the p-NA group. The CD spectra indicate that Boc-Gly-DeltaAla-Gly-Phe-p-NA and Gly-DeltaAla-Gly-Phe-p-NA.TFA are unordered, independently of the solvent.     

Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dehydrophenylalanine zippers: strong helix-helix clamping through a network of weak interactions

A decapeptide Boc-L-Ala-(Delta Delta Phe)(4)-L-Ala-(Delta Delta Phe)3-Gly-OMe (Peptide I) was synthesized to study the preferred screw sense of consecutive alpha,beta-dehydrophenylalanine (Delta Delta Phe) residues. Crystallographic and CD studies suggest that, despite the presence of two L-Ala residues in the sequence, the decapeptide does not have a preferred screw sense. The peptide crystallizes with two conformers per asymmetric unit, one of them a slightly distorted right-handed 3(10)-helix (X) and the other a left-handed 3(10)-helix (Y) with X and Y being antiparallel to each other. An unanticipated and interesting observation is that in the solid state, the two shape-complement molecules self-assemble and interact with an extensive network of C-H...O hydrogen bonds and pi-pi interactions, directed laterally to the helix axis with amazing regularity. Here, we present an atomic resolution picture of the weak interaction mediated mutual recognition of two secondary structural elements and its possible implication in understanding the specific folding of the hydrophobic core of globular proteins and exploitation in future work on de novo design.     

Re-engineering monovalent cation binding sites of methylamine dehydrogenase: effects on spectral properties and gated electron transfer

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ)-dependent enzyme that catalyzes the oxidative deamination of primary amines. Monovalent cations are known to affect the spectral properties of MADH and to influence the rate of the gated electron transfer (ET) reaction from substrate-reduced MADH to amicyanin. Two putative monovalent cation binding sites in MADH have been identified by X-ray crystallography [Labesse, G., Ferrari, D., Chen, Z.-W., Rossi, G.-L., Kuusk, V., McIntire, W. S., and Mathews, F. S. (1998) J. Biol. Chem. 273, 25703-25712]. One requires cation-pi interactions involving residue alpha Phe55. An alpha F55A mutation differentially affects these two monovalent cation-dependent phenomena. The apparent K(d) associated with spectral perturbations increases 10-fold. The apparent K(d) associated with enhancement of the gated ET reaction becomes too small to measure, indicating that either it has decreased more than 1000-fold or the mutation has caused a conformational change that eliminates the requirement for the cation for the gated ET. These results show that of the two binding sites revealed in the structure, cation binding to the distal site, which is stabilized by the cation-pi interactions, is responsible for the spectral perturbations. Cation binding to the proximal site, which is stabilized by several oxygen ligands, is responsible for the enhancement of the rate of gated ET. Another site-directed mutant, alpha F55E MADH, exhibited cation binding properties that were the same as those of the native enzyme, indicating that interactions with the carboxylate of Glu can effectively replace the cation-pi interactions with Phe in stabilizing monovalent cation binding to the distal site.