Specific inhibitory conformation of dipeptides for chymotrypsin

Based on the analyzed conformation of a chymotrypsin inhibitor H-delta Phe-Phe-OMe, we have designed a series of diastereomeric phenylalanylphenylalanine methyl esters and derivatives as possible inhibitors. Among the peptides synthesized, H-D-Phe-L-Phe-OMe was found to be very resistant to chymotrypsin in spite of its L-Phe-OMe structure at the C-terminus. It inhibited the enzyme fairly strongly and competitively with Ki = 9.0 x 10(-5) M in the assay using Ac-Tyr-OEt as a substrate. The measurements of the NOEs in high-resolution 1H-NMR analyses indicated the presence of the hydrophobic core built by the intramolecular interaction between the D-Phe-phenyl and ester-methyl groups. It was suggested that this core interacts with the chymotrypsin S2 site (Trp215) and Phe2 with the S1 site. The backbone structure of this dipeptide was assumed to be in an inhibitory conformation that fits the active center of the enzyme.     

Alpha helix capping in synthetic model peptides by reciprocal side chain–main chain interactions: Evidence for an N terminal “capping box”

A significant fraction of the amino acids in proteins are alpha helical in conformation. Alpha helices in globular proteins are short, with an average length of about twelve residues, so that residues at the ends of helices make up an important fraction of all helical residues. In the middle of a helix, H-bonds connect the NH and CO groups of each residue to partners four residues along the chain. At the ends of a helix, the H-bond potential of the main chain remains unfulfilled, and helix capping interactions involving bonds from polar side chains to the NH or CO of the backbone have been proposed and detected. In a study of synthetic helical peptides, we have found that the sequence Ser-Glu-Asp-Glu stabilizes the alpha helix in a series of helical peptides with consensus sequences. Following the report by Harper and Rose, which identifies SerXaaXaaGlu as a member of a class of common motifs at the N termini of alpha helices in proteins that they refer to as “capping boxes,” we have reexamined the side chain–main chain interactions in a varient sequence using ¹H NMR, and find that the postulated reciprocal side chain-backbone bonding between the first Ser and last Glu side chains and their peptide NH partners can be resolved: Deletion of two residues N terminal to the Ser-Glu-Asp-Glu sequence in these peptides has no effect on the initiation of helical structure, as defined by two-dimensional (2D) NMR experiments on this variant. Thus the capping box sequence Ser-Glu-Asp-Glu inhibits N terminal fraying of the N terminus of alpha helix in these peptides, and shows the side chain–main chain interactions proposed by Harper and Rose. It thus acts as a helix initiating signal. Since normal a helix cannot propagate beyond the N terminus of this structure, the box acts as a termination signal in this direction as well. © 1994 John Wiley & Sons, Inc.     

Influence of hydrogen bonding,main chain–side chain interactions,and protecting groups on the excimer formation of bis(pyrenylalanine) peptides

Dipeptides of the aromatic fluorescent amino acid, pyrenylalanine, are studied using both stationary and transient fluorescence techniques. Since the conformational transitions of the peptide chain are slow compared to the decay of the pyrene excited state, both ground state conformations, adopted by the peptide, i.e., C₅ and C₇, can be monitored separately. Kinetic models are proposed to describe the molecular dynamics of the peptide chain as probed by the intramolecular excimer formation between both pyrene chromophores. These kinetic schemes explain the influence of solvent, chain chirality, main chain–side chain interactions, and nature of the protecting groups on the emission spectrum and the fluorescence decay profile of these model peptides. These schemes also provide a tool to calculate rate constants of conformational transitions and excimer formation. By comparing the kinetic and thermodynamic parameters of the various compounds, the influence of a structural modification on the molecular dynamics of the peptide chain is determined.     

Highly potent side chain–main chain cyclized dermorphin–deltorphin analogues: An integrated approach including synthesis,bioassays, NMR spectroscopy and molecular modelling

Our continuing efforts to study structure–activity relationships of peptide opioids have resulted in the synthesis of a series of cyclic opioids related to dermorphins and deltorphins. The biological activities of the compounds have been determined and the conformational analyses carried out using ¹H-NMR spectroscopy and molecular modelling. The three compounds in the series Tyr-c[D -Orn-Phe-Ala], Tyr-c[D -Lys-Phe-Ala], and Tyr-c[A₂Bu-Phe-Ala-Leu] are cyclized via a lactam bridge from the side-chain of the residue at the second position with the carboxyl terminus of each compound. The molecules incorporate 12-, 13- and 14-membered rings, respectively. They include a phenylalanine at the third position which is a distinguishing characteristic of dermorphins and deltorphins. The guinea pig ileum and mouse vas deferens assays show that the compounds are highly active at both μ- and δ-opioid receptors. The compounds are all highly effective antinociceptive agents as measured by the intrathecal rat hot plate test. Conformational analyses of the molecules indicate that they can adopt topochemical arrays required for bioactivity at both μ- and δ-receptors which explains their high activity in both guinea pig ileum and mouse vas deferens in vitro assays. The results support our models for μ- and δ-receptor activity for constrained peptide opioids.     

Backbone side chain interactions in peptides. I. Crystal structures of model dipeptides with the Pro-Ser sequence

The preferential occurrence of amino-acid residues having short polar side-chain within beta-folded regions of crystallized proteins suggests the existence of some stabilizing interaction involving the side polar function. Three model dipeptides tBuCO-L-Pro-L-Ser-NHMe 1, tBuCO-L-Pro-D-Ser-NHMe 2 in the pure enantiomeric a and racemic b forms, and iPrCO-L-Pro-D-Ser-OMe 3 have been investigated in the solid state by X-ray crystallography. Homo and heterochiral sequences 1 and 2 are folded in the beta I and beta II types, respectively, whereas 3 obviously accommodates an open conformation. Besides the i + 3 leads to i hydrogen bond typical of beta-bends in 1, 2a, and 2b, the Ser NH group in all four crystal structures is a proton donor to the lone orbitals of the Ser O gamma oxygen atom. The result is that the disposition of the Ser C alpha--C beta bond corresponds to the rotamer III (chi 1 congruent to 60 degrees). As shown by the crystal structure of 3, the intra-Ser NH. . .O gamma hydrogen bonding is not restricted to beta-folded Pro-Ser sequences. Therefore, this interaction is not only a stabilizing factor for beta-turns but it is also probably responsible for the already mentioned stability of rotamer III for the Ser C alpha--C beta bond in peptides and protein.     

Effect of residual side chain blocking groups and side chain charge on polypeptide conformation in aqueous solution

The circular dichroism (CD) spectrum of poly-L-lysine and poly-L -glutamic acid has been investigated in the presence of a small percent of side-chain blocking groups. The blocking groups benzyl, methyl, and carbobenzoxy show qualitatively similar effects. Less than five mole percent of aromatic blocking groups alters the CD spectrum. Consequently, unsuspected blocking groups may account for the variation observed in CD spectra of these polymers. A weak CD band at 235–240 nm was observed for the disordered unblocked polymer even in the absence of electrolyte. Viscosity data indicate that in salt-free solutions these chains at neutral pH still behave as random coils though with reduced conformational freedom, in contrast to some polyelectrolytes which behave as rigid rods in the absence of electrolyte. The viscosity data bring into question the relevance of isolated molecule conformational calculations to experimental CD spectra.     

Mode matches in hydrophobic free energy eigenfunctions predict peptide–protein interactions

The dominant statistical hydrophobic free energy inverse frequencies, amino acid wavelengths as hydrophobic modes, of neurotensin (NT), cholescystokinin (CCK), the human dopamine D₂ receptor [(DA)D₂], and the human dopamine transporter (DAT) were determined using orthogonal decomposition of the autocovariance matrices of their amino acid sequences as hydrophobic free energy equivalents in kcal/mol. The leading eigenvalues-associated eigenvectors were convolved with the original series to construct eigenfunctions. Eigenfunctions were further analyzed using discrete trigonometric wavelet and all poles, maximum entropy power spectral transformations. This yielded clean representations of the dominant hydrophobic free energy modes, most of which are otherwise lost in the smoothing of hydropathy plots or contaminated by end effects and multimodality in conventional Fourier transformations. Mode matches were found between NT and (DA)D₂ and between CCK and DAT, but not the converse. These mode matches successfully predicted the nonlinear kinetic interactions of NT-(DA)D₂ in contrast with CCK-(DA)D₂ on ³H-spiperone binding to (DA)D₂, and by CCK-DAT but not NT-DAT on [N-methyl-³H]-WIN 35,428 binding to DAT in (DA)D₂ and DAT cDNA stably transfected cell lines without known NT or CCK receptors. Computation of the dominant modes of hydrophobic free energy eigenfunctions may help predict functionally relevant peptide–membrane protein interactions, even across neurotransmitter families. © 1998 John Wiley & Sons, Inc. Biopoly 46: 89–101, 1998     

Influence of electrostatic interactions on the sugar–phosphate backbone conformation in DNA

The influence of sugar ring flexibility in DNA on the mechanism of the B ? A conformational transition is studied. The dipole moment of the deoxyribose as a function of its puckered states is calculated by the quantum-mechanical method using the MINDO/3 approximation. The interaction of the sugar dipole with the neighbor molecular groups in polynucleotide chains is estimated. The sugar dipole interaction with phosphate groups and counterions is shown to be strong and capable to deform the pseudorotation potential of deoxyribose. The effective pseudorotation potential of sugar ring in the B and A helices is obtained. The results are used to explain the behavior of Raman bands in the region of sugar–phosphate vibrations. The mechanism of the effect of electrostatic forces on the sugar–phosphate backbone conformation, which is essential for the B ? A and other structure transitions, is offered. © 1993 John Wiley & Sons, Inc.     

CIDNP study of the aromatic side chain interactions in myotoxina

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxina, aCrotalus venom toxin which acts as blocker of the Ca²⁺ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr₁, His₅, His₁₀, and (possibly) F₁₂. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr, is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr₁, and proton 4 in each of His₅ and His₁₀ are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca²⁺ ATPase.     

Binding of cationic pentapeptides with modified side chain lengths to negatively charged lipid membranes: Complex interplay of electrostatic and hydrophobic interactions

Basic amino acids play a key role in the binding of membrane associated proteins to negatively charged membranes. However, side chains of basic amino acids like lysine do not only provide a positive charge, but also a flexible hydrocarbon spacer that enables hydrophobic interactions. We studied the influence of hydrophobic contributions to the binding by varying the side chain length of pentapeptides with ammonium groups starting with lysine to lysine analogs with shorter side chains, namely ornithine (Orn), α,γ-diaminobutyric acid (Dab) and α, β-diaminopropionic acid (Dap). The binding to negatively charged phosphatidylglycerol (PG) membranes was investigated by calorimetry, FT-infrared spectroscopy (FT-IR) and monolayer techniques. The binding was influenced by counteracting and sometimes compensating contributions. The influence of the bound peptides on the lipid phase behavior depends on the length of the peptide side chains. Isothermal titration calorimetry (ITC) experiments showed exothermic and endothermic effects compensating to a different extent as a function of side chain length. The increase in lipid phase transition temperature was more significant for peptides with shorter side chains. FTIR-spectroscopy revealed changes in hydration of the lipid bilayer interface after peptide binding. Using monolayer techniques, the contributions of electrostatic and hydrophobic effects could clearly be observed. Peptides with short side chains induced a pronounced decrease in surface pressure of PG monolayers whereas peptides with additional hydrophobic interactions decreased the surface pressure much less or even lead to an increase, indicating insertion of the hydrophobic part of the side chain into the lipid monolayer.     

Influence of hydrophobic amino acid residues on the esterification of dipeptides by papain

Summary Esterification of dipeptide was performed with papain immobilized on XAD-7 with methylene chloride as the organic solvent. The esterification of a peptide substrate Box-X-Y-OH is promoted by the presence of a hydrophobic aminoacid residue (e.g., Phe, Leu) in the X (P2) position. Good yields were observed for example with Boc-Phe-Glu-OH and Boc-Ala-Glu-OH. With a hydrophilic aminoacid residue in the X position no esterification of the dipeptide takes place. Instead hydrolysis of the peptide bond occurs. This is also the case with glycine as the aminoacid; for instance Boc-Leu-Gly-OH gives a 80% yield of esterification whereas Boc-Gly-Leu-OH gives only the aminoester Boc-Gly-OR.     

Conformationally restricted analogs of oxytocin; stabilization of inhibitory conformation

Analogs of oxytocin containing tetrahydroisoquinoline carboxylic acid (Tic) of L or D configuration in position 2 were synthesized and their biological activities were tested. Both analogs showed negligible agonist activity in uterotonic, galactogogic, and pressor assays, but they are in vitro uterotonic inhibitors. In comparison with oxytocin analogs containing L- or D-phenylalanine in position 2, the analog with the D-configuration of the conformationally fixed aromatic residue has significantly increased inhibitory activity which suggests that the proper conformation for the interaction with the receptor, but not for its activation, was stabilized. 1H NMR and CD studies, supported by theoretical calculations, suggest that the conformational properties of the analog containing D-tetrahydroisoquinoline carboxylic acid are similar to those of [2-D-phenylalanine]oxytocin.     

The conformation of the lysyl side chain of substrates at the active center of trypsin

In order to study the conformation of the side chain of lysine substrates bound to the active center of trypsin, two lysine analogs, cis- and trans-2,6-diamino-4-hexenoic acids (4,5-dehydrolysines) were synthesized and kinetic parameters for the hydrolysis of benzoyl methyl esters and phenylthiazolones of these analogs by this enzyme were compared with those of the corresponding lysine derivatives. The derivatives of cis-4,5-dehydrolysine were hydrolyzed much more slowly than those of lysine, owing largely to the small kcat values for the former. On the other hand, the derivatives of the trans-isomer were hydrolyzed at about the same rates as those of lysine and the values of both Km and kcat of the former are also similar to those of the latter. These results indicate that the conformation of the side chain of the lysine derivatives hydrolyzed by trypsin is such that the beta- and epsilon-carbons are in a trans-like conformation, as suggested by X-ray crystallographic studies of inhibitor-trypsin complex.     

Compatibility of the conformationally rigid CF3-Bpg side chain with the hydrophobic coiled-coil interface

3-(Trifluoromethyl)bicyclopent-[1.1.1]-1-yl glycine (CF₃-Bpg) has previously been established as a useful ¹⁹F NMR label to analyse the structures of oligomeric membrane-active peptides or transmembrane segments. To systematically examine the effect of side chain volume, conformational rigidity, and hydrophobicity of CF₃-Bpg in polypeptide environments the amino acid was incorporated into an established coiled-coil based screening system. A single substitution of either valine (position a16) or leucine (position d19) within the hydrophobic core of the heteromeric coiled coil has practically no effect on its structure. Despite its comparatively high hydrophobicity, however, the stiff and bulky side chain of CF₃-Bpg is not so well accommodated by the hydrophobic core as it leads to a more pronounced destabilization than observed for other, more polar fluorinated amino acids which carry more flexible side chains. CF₃-Bpg is therefore a useful ¹⁹F NMR label, though not for monitoring the stability of such helix–helix interactions.     

Evaluation of a novel bifunctional xylanase–cellulase constructed by gene fusion

An artificial bifunctional enzyme, xylanase–cellulase, has been prepared by gene fusion. Three chimeric genes were constructed that encoded fusion proteins of different lengths. The fusion proteins exhibited both xylanase (XynX) and cellulase (Cel5Z::Ω) activity when cel5Z::Ω was fused downstream of xynX, but not when xynX was fused downstream of cel5Z::Ω. Activities of bifunctional enzymes decreased when a shorter xylanase peptide was fused. Three fusion enzymes were purified, and the molecular weights of the enzymes were estimated by CMC-SDS-PAGE and XYN-SDS-PAGE to be 149, 129, and 87 kDa, respectively. The fusion enzymes displayed optimum cellulase activity at pH 8.0 and 50 °C and optimum xylanase activity at pH 8.0 and 70 °C.     

CIDNP study of the aromatic side chain interactions in myotoxina

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxina, aCrotalus venom toxin which acts as blocker of the Ca²⁺ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr₁, His₅, His₁₀, and (possibly) F₁₂. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr, is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr₁, and proton 4 in each of His₅ and His₁₀ are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca²⁺ ATPase.     

Effect of lysine side chain length on intra-helical glutamate--lysine ion pairing interactions

Ion-pairing interactions are important for protein stabilization. Despite the apparent electrostatic nature of these interactions, natural positively charged amino acids Lys and Arg have multiple methylenes linking the charged functionality to the backbone. Interestingly, the amino acids Lys and Orn have positively charged side chains that differ by only one methylene. However, only Lys is encoded and incorporated into proteins. To investigate the effect of side chain length of Lys on ion-pairing interactions, a series of 12 monomeric alpha-helical peptides containing potential Glu-Xaa (i, i+3), (i, i+4) and (i, i+5) (Xaa = Lys, Orn, Dab, Dap) interactions were studied by circular dichroism (CD) spectroscopy at pH 7 and 2. At pH 7, no Glu-Xaa (i, i+5) interaction was observed, regardless of the Xaa side chain length. Furthermore, only Lys was capable of supporting Glu-Xaa (i, i+3) interactions, whereas any Xaa side chain length supported Glu-Xaa (i, i+4) interactions. Side chain conformational analysis by molecular mechanics calculations showed that the side chain length of Lys enables the Glu-Xaa (i, i+3) interaction with lower energy conformations compared to residues with side chain lengths shorter than that of Lys. Furthermore, these calculated low energy conformers were consistent with conformations of intra-helical Glu-Lys salt bridges in a non-redundant protein structure database. Importantly, the CD spectra for peptides with Glu-Lys interactions did not alter significantly upon changing the pH because of a greater contribution to these interactions by forces other than electrostatics. Incorporating side chains just one methylene shorter (Orn) resulted in significant pH dependence or lack of interaction, suggesting that nature has chosen Lys to form durable interactions with negatively charged functional groups.     

The Gram-positive side of plant–microbe interactions

Plant growth and development are significantly influenced by the presence and activity of microorganisms. To date, the best-studied plant-interacting microbes are Gram-negative bacteria, but many representatives of both the high and low G+C Gram-positives have excellent biocontrol, plant growth-promoting and bioremediation activities. Moreover, actinorhizal symbioses largely contribute to the global biological nitrogen fixation and many Gram-positive bacteria promote other types of symbioses in tripartite interactions. Finally, several prominent and devastating phytopathogens are Gram-positive. We summarize the present knowledge of the beneficial and detrimental interactions of Gram-positive bacteria with plants to underline the importance of this particular group of bacteria.