Leu Peptide Research Articles

Phospholipid Membrane Interactions of Model Ac-WL-X-LL-OH Peptides Investigated by Solid-State Nuclear Magnetic Resonance.

The role of aromatic amino acids in peripheral protein membrane binding has been reported to involve cation-π interactions with choline lipids. In this study, we have investigated the interactions of the model pentapeptide Ac-WL-X-LL-OH (where X = L, Y, F, or W) with the phospholipid membrane using solid-state NMR. The effect of guest residue X on the peptide-lipid interactome was complementary to the seminal report on the interfacial hydrophobicity scale by Wimley and White. We found that the phospholipids retained a lamellar phase in the presence of each of the peptides with an aromatic X residue, whereas the Leu peptide perturbed the bilayer to an extent where an additional isotropic phase was observed. The solid-state NMR 13C and 31P data provide additional information on the influence of these short peptides on the membrane that has not been previously reported. The magnitude of membrane perturbation was in the order of guest residue X = L > Y~F > W, which is consistent with the relative amino acid interfacial affinity reported by Wimley and White. Further work is, however, required to uncover the behavior of the peptide and localization in the membrane domain due to ambiguity of the 13C NMR data. We have launched efforts in this regard for the objective of better understanding the role of aromatic amino acids in peripheral membrane protein binding.

Asymmetric 1,4-Addition Reactions Catalyzed by N-Terminal Thiourea-Modified Helical l-Leu Peptide with Cyclic Amino Acids.

N-terminal thiourea-modified l-Leu-based peptide {(3,5-diCF3 Ph)NHC(=S)-(l-Leu-l-Leu-Ac5 c)2 -OMe} with five-membered ring α,α-disubstituted α-amino acids (Ac5 c) catalyzed a highly enantioselective 1,4-addition reaction between β-nitrostyrene and dimethyl malonate. The enantioselective reaction required only 0.5 mol % chiral peptide-catalyst in the presence of i Pr2 EtN (2.5 equiv.), and gave a 1,4-adduct with 93 % ee of an 85 % yield. As Michael acceptors, various β-nitrostyrene derivatives such as methyl, p-fluoro, p-bromo, and p-methoxy substituents on the phenyl group, 2-furyl, 2-thiophenyl, and naphthyl β-nitroethylenes could be applied. Furthermore, various alkyl malonates and cyclic β-keto-esters could be used as Michael donors. It became clear that the length of the peptide chain, a right-handed helical structure, amide N-Hs, and the N-terminal thiourea moiety play crucial roles in asymmetric induction.

Regulation of the expression of key signalling molecules in mTOR pathway of skeletal muscle satellite cells in neonatal chicks: Effects of leucine and glycine-leucine peptide.

This study was conducted to analyse the effects of leucine (Leu) and glycine (Gly)-Leu peptide on expressions of key signalling molecules in mTOR pathway of skeletal muscle satellite cells in neonatal chicks. The 4-day-old male AA broilers with similar weight were selected to obtain the broiler skeletal muscle satellite cells with the two-step method of collagenase-I and trypsin digestion. The satellite cells were subjected to primary culture in vitro, and they were cultured in DMEM medium with the Leu concentration of 0.2mM and 2mM as well as with the Gly-Leu peptide concentration of 0.2mM and 2mM. The experiment lasted for 5days. The results showed that TOR, S6K1 and 4E-BP1 mRNA expressions in the medium with Leu concentration of 2mM were significantly higher than that in 0.2mM group (p<0.05). There was no difference between the medium with Gly-Leu concentration of 2mM and 0.2mM on the TOR, S6K1 and 4E-BP1 mRNA expressions (p>0.05). In conclusion, Leu significantly increases TOR, S6K1 and 4E-BP1 mRNA expressions of skeletal muscle satellite cells, but Gly-Leu peptide has no effect on them.

Relative role(s) of leucine versus isoleucine in the folding of membrane proteins

AbstractLarge, hydrophobic residues (isoleucine, leucine, and valine) dominate sequences of transmembrane (TM) helices in membrane proteins (total ∼34%), but their relative roles in mediating the biologically relevant protein–lipid and protein–protein interactions have not been systematically evaluated. Here we have synthesized Leu‐containing Lys‐tagged hydrophobic peptides of identical composition, where sequences have been designed with their Leu residues either scrambled (sequence KKKLAASALAAAWLAALALSAAKKK); clustered (KKKAAASAALLLWLLAAAASAAKKK); or “lipopathic” (all Leu on one helical face) (KKKAAASLAALLWALLAAASAAKKK). These peptides were compared by several biophysical/biochemical techniques to the corresponding set of peptides where the Leu residues are replaced by the isosteric Ile residues. Circular dichroism spectra showed that all peptides were helical in POPC liposomes, as confirmed by blue shifts in Trp fluorescence spectra, notably with the Ile‐lipopathic peptide displaying increased Trp burial versus its Leu counterpart. Quenching experiments with a dibromo‐PC lipid indicated deeper membrane penetration of the Ile versus the Leu lipopathic peptide—a result supported by protease degradation assays where Ile peptides reconstituted into lipid bilayers were significantly more protected from the protease than the Leu peptides. Assessment of Trp blue shifts in the presence of lipid bilayers of varied lipid packing indicated that Leu/Ile peptide interactions are dependent on lipid composition. The overall results suggest that two main interactions tend to dominate Leu and Ile interactions within the membrane: (1) hydrophobic interactions between amino acid side chains and the surrounding lipid; and (2) degree of disruption of lipid–lipid packing. This “battle of giants” likely underlies the specific role(s) that Leu and Ile will play in the folding of a given membrane protein.

Agricultural potential of rhizospheric Bacillus subtilis strains exhibiting varied efficiency of surfactin production

Six Bacillus subtilis strains collected from broad-leaved trees rhizosphere and exhibiting an ability of surfactin production were investigated as potent plant biofertilizers with biocontrol activity. Lipopeptide (LP) biosurfactants were extracted by a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) procedure proposed as a quick and effective method of isolating LP biosurfactants. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF) was applied to detect the lipopeptides. Predominant peaks at m/z 1016, 1030, 1044, 1058 and 1072 were putatively assigned as surfactin Na adducts possess a Glu-Leu-Leu-Val-Asp-Leu–Leu peptide and the β-OH fatty acid with a different length (C13-C16) whereas m/z 1016 was C13 surfactin Na adduct with Val in position 7 on the peptide moiety. Four other ions m/z 1051, 1065, 1079 and 1093 were recognized as iturin A Na adducts. Five fengycin homologues with Na adducts (m/z 1471, 1485, 1499, 1513 and 1527) were also determined. The quantitative analyses of the compounds were performed with the use of liquid chromatography-mass spectrometry (LC–MS/MS). Strains IM 7, IM 16 and IM 17 cultivated in Luria-Bertani medium for 48h produced surfactin alone at the concentrations below 10mgl−1. In contrast, strains KP7, IM 13 and IM 14 produced surfactin much more efficiently. Surfactin concentrations in 72-h-old cultures reached 100.3, 55.3 and 124.4mgl−1, respectively and were accompanied by the production of iturin in concentrations 5.7, 142.5 and 2.3mgl−1, respectively. Additionally, in Cooper’s medium, strain KP7 increased twofold the production of surfactin and iturin. Only surfactin overproducers (KP7, IM 13 and IM 14) exhibited swarming motility and formed biofilms, limited the growth of Botrytis cinerea and Aspergillus flavus strains, produced cellulases and inulinases and enhanced cucumber seed germination in vitro. All examined surfactin overproducers produced siderophores and, additionally, KP7 and IM 14 solubilized phosphates. Among all examined surfactin overproducers only IM 13 during the growth in the presence of F. oxysporum mycelium (in dual liquid culture) increased about twofold surfactin and iturin secretion and almost completely suppressed fungal biomass growth. The obtained results revealed that the studied surfactin overproducers were equipped with various plant growth-promoting (PGP) traits allowing them to promote plant development in a diverse manner.

DapE can function as an aspartyl peptidase in the presence of Mn2+.

Extracts of a multiply peptidase-deficient (pepNABDPQTE iadA iaaA) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity that is dependent on Mn(2+). Purification of this activity followed by N-terminal sequencing of the protein suggested that the Mn(2+)-dependent peptidase is DapE (N-succinyl-L,L-diaminopimelate desuccinylase). A dapE chromosomal disruption was constructed and transduced into a multiply peptidase-deficient (MPD) strain. Crude extracts of this strain showed no aspartyl peptidase activity, and the strain failed to utilize Asp-Leu as a leucine source. The dapE gene was cloned into expression vectors in order to overproduce either the native protein (DapE) or a hexahistidine fusion protein (DapE-His(6)). Extracts of a strain carrying the plasmid overexpresssing native DapE in the MPD dapE background showed a 3,200-fold elevation of Mn(2+)-dependent aspartyl peptidase activity relative to the MPD dapE(+) strain. In addition, purified DapE-His(6) exhibited Mn(2+)-dependent peptidase activity toward aspartyl dipeptides. Growth of the MPD strain carrying a single genomic copy of dapE on Asp-Leu as a Leu source was slow but detectable. Overproduction of DapE in the MPD dapE strain allowed growth on Asp-Leu at a much faster rate. DapE was found to be specific for N-terminal aspartyl dipeptides: no N-terminal Glu, Met, or Leu peptides were hydrolyzed, nor were any peptides containing more than two amino acids. DapE is known to bind two divalent cations: one with high affinity and the other with lower affinity. Our data indicate that the form of DapE active as a peptidase contains Zn(2+) in the high-affinity site and Mn(2+) in the low-affinity site.

Context-dependent conformation of diethylglycine residues in peptides.

Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C5) or helical conformations. The conformations of three tetrapeptides Boc-Xxx-Deg-Xxx-Deg-OMe (Xxx=Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C5 conformation. NMR evidence supports a folded beta-turn conformation involving Deg(2)-Gly(3), stabilized by a 4-->1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)-Deg(2) adopting a distorted type II/II' conformation, while the Deg(2)-Pro(3) segment adopts a type III/III' structure. A lone water molecule is inserted into the potential 4-->1 hydrogen bond of the Gly(1)-Deg(2) beta-turn.

Chromatographic resolution of tryptophan enantiomers with l-Leu– l-Leu– l-Leu peptide : Effects of mobile phase composition and chromatographic support

Tryptophan enantiomers have been separated by zwitterion pair chromatography using l-leucine– l-leucine– l-leucine peptide as the zwitterion pairing agent. The peptide ligand is adsorbed onto an octadecylsilane support with excess ligand present in bulk solution. This article examines the roles of the hydrophobic matrix and the mobile phase components on tryptophan enantiomer binding and resolution. Capacity factors and enantioselectivites are given for both hydrophobic and hydrophilic matrices using mobile phases containing Leu–Leu–Leu peptide and/or salt. A decrease in selectivity upon the addition of mobile phase salt suggests that quadrupolar ion-pairing contributes to chiral recognition. Results indicate that binding is significantly reduced and separation is not achieved when Leu–Leu–Leu is coupled onto cross-linked or polymerized hydrophilic resins as well as onto macroporous polystyrene resin. However, resin-immobilized Leu–Leu–Asp–Leu–Leu–Leu, Leu–Leu–Glu–Leu–Leu–Leu, and Leu–Leu–Leu–Glu–Leu–Leu peptides, with ion-pairing sites designed to mimic the Leu–Leu–Leu-saturated C 18 support, also do not resolve tryptophan enantiomers. This suggests the Leu–Leu–Leu structure is critical for enantiomer resolution. Because d- and l-tryptophan are separated in the absence of bulk Leu–Leu–Leu, chiral discrimination is believed to occur at the surface of the octadecylsilane support.

Design of membrane-inserting peptides: spectroscopic characterization with and without lipid bilayers.

This paper reports the spectroscopic characterization of two de novo peptides. The first sequence, Ala peptide = H2N-Ala27-Tyr-Lys6-CONH2, gives circular dichroism (CD) and Fourier transform infrared (FTIR) spectra characteristic of beta structure in solution, binds to lipid bilayer vesicles poorly, and tends to precipitate in buffered 0.1 M salt solutions. In the second sequence, Leu peptide = H2N-Ala2-Leu3-Ala22-Tyr-Lys6-CONH2, three leucines are substituted for three alanine residues. This small sequence change results in CD spectra that are characteristic of helical structures, while the FTIR spectra give evidence for complex equilibria between multiple structures in solution. The Leu peptide does not precipitate in buffered salt solutions and binds to lipid bilayers. The polarized attenuated total reflectance infrared spectra provide evidence of a transmembrane orientation for the helical peptide in lipid bilayers. The collective spectroscopic results are summarized in a tentative model in which the Leu peptide exhibits multiple equilibria between extended unordered, helix, and coiled-coil structures in solution; when lipid vesicles are added, the peptide binds to the lipid surface and then inserts into the lipid in a transmembrane orientation. The slow kinetics exhibited by the peptide suggest multiple conformational changes during the lipid-peptide interactions. The design rationale for the peptides is included in an appendix.

Reversed-phase chromatography of synthetic amphipathic α-helical peptides as a model for ligand/receptor interactions Effect of changing hydrophobic environment on the relative hydrophilicity/hydrophobicity of amino acid side-chains

To mimic a hydrophobic protein binding domain, which is a region on the surface of a protein that has a preference or a specificity to interact with a complementary surface, we have designed amphipathic α-helical peptides where the non-polar face interacts with the non-polar surface of a reversed-phase stationary phase. Two series of potentially amphipathic α-helical peptides, a native Ala peptide (AA9) and a native Leu peptide (LL9), were designed where the native peptide contains 7 residues of either Ala or Leu, respectively, in its non-polar face. This design results in an overall hydrophobicity of the non-polar face of the Leu peptide that is greater than that of the non-polar face of the native Ala peptide. Mutants of the native Ala-face peptide, AX9, and the native Leu-face peptide, LX9, were designed by replacing one residue in the centre of the non-polar face in both series of peptides. Therefore, by changing the hydrophobicity of the environment surrounding the mutated amino acid side-chain, the effect on the hydrophilicity/hydrophobicity of each amino acid side-chain could be determined. Using the substitutions Ala, Leu, Lys and Glu, it was shown that the maximum hydrophilicity of these amino acid side-chains could be determined when the environment surrounding the mutation is maximally hydrophobic; whereas its maximum hydrophobicity can be determined when the environment surrounding the mutation is minimally hydrophobic. This procedure was further extended to the remaining amino acids commonly found in proteins and it was determined that this general principle applies to all 20 amino acids. These results have major implications to understanding the hydrophilicity/hydrophobicity of amino acid side-chains and the role side-chains play in the folding and stability of proteins.

Substitutions at the P 2′ site of gag p17–p24 affect cleavage efficiency by HIV-1 protease

Heptapeptide substrates containing a single amino acid substitution at the p2′ position of the gag p17–p24 junction (S-Q-N-Y-P-X-V where X = G, A, L, I, F, and W) were compared as substrates for HIV-1 protease. Binding of the Ile-, Leu-, and Ala- containing peptides was about equal although hydrolysis was 20-fold lower for the Ala and Leu peptides compared to Ile. Insertion of Gly or Phe at the p2′ position resulted in significantly lower cleavage of the peptide while a Trp-containing peptide was not cleaved. These data suggest that a relatively small hydrophobic amino acid is important for hydrolysis and binding at this site. Structure-activity studies such as those described here may be useful in the design of specific inhibitors for HIV-1 protease.

Characterization of the small hydrophobic proteins associated with pulmonary surfactant

Lipid extracts of bovine pulmonary surfactant, which retain many of the biophysical characteristics of natural surfactant, contain approx. 98% lipid and 2% protein, as determined by amino acid analysis. Polyacry (amide/urea gel electrophoresis reveals that lipid extract surfactant contained a major apoprotein band with apparent M r 3500 and minor apoprotein bands with apparent M r 15000 and 7000. After reduction, the 15 kDa band disappears and is replaced by a prominent band with apparent M r = 5000. Reduction also results in a relative diminution of the 7 kDa band and a relative increase in the intensity of the 3.5-kDa band. Edman degradation reveals two major peptide sequences which have been designated surfactant-associated peptide (N-terminal Phe) and surfactant-associated peptide (N-terminal Leu) and a minor sequence designated surfactant-associated peptide (N-terminal Ile). The latter surfactant-associated peptide appears to be related to the N-terminal Leu peptide but lacks the terminal Leu. N-Terminal analysis by dansylation demonstrates that the 15 and 5 kDa (reduced) apoprotein species contain N-terminal Phe, Leu and Ile. The 3.5 and 7 kDa bands contain only N-terminal Leu and Ile. Chromatography of lipid extracts on silicic acid columns gives rise to fraction I, which contains protein and phosphatidylglycerol, and fraction II, which contains protein, phosphatidylglycerol and phosphatidylethanolamine. Fraction I was primarily composed of the 15-kDa apoproteins, while fraction II contained mainly the 3.5 and 7 kDa apoproteins. Both fractions exhibited biophysical activity after reconstitution with dipalmitoylphosphatidylcholine. These results indicate that lipid extracts contain an oligomer of 15 kDa containing surfactant-associated peptide (N-terminal Phe) and surfactant-associated peptides (N-terminal Leu or Ile) which interact through sulfhydryl and perhaps other bonds. Lipid extracts also contain 3.5 kDa monomers of surfactant-associated peptides with N-terminal Leu and N-terminal He which can dimerize through sulfhydryl and perhaps hydrophobic interactions.

Relationship between α-helical propensity and formation of the ribonuclease-S complex

Variant semisynthetic ribonuclease-S complexes were characterized in which the helical glutamic acid 9 residue was replaced by either leucine or glycine. The Leu-9 and Gly-9 synthetic peptides, corresponding otherwise to residues 1 through 15 of bovine pancreatic ribonuclease, were studied with respect to the ability to bind, and generate enzymic activity, with the complementary native protein fragment containing residues 21 through 124 of ribonuclease (RNAase-S-(21–124)). Both the Leu and Gly peptides bind to the RNAase-S-(21–124) to yield complexes with catalytic properties similar to those obtained with the Glu-9-containing peptide of residues 1 through 20 of ribonuclease (RNAase-S-(1–20)). However, whereas the binding affinity of Leu peptide to RNAase-S-(21–124) is only a factor of three less than that for RNAase-S-(1–20), that for Gly peptide is about 20-fold less than that for RNAase-S-(1–20). The stronger binding of Leu than Gly peptide corresponds to the observed propensity of leucine but not glycine for the α-helical conformation in globular proteins. In spite of the weakened affinity of the Gly peptide for RNAase-S-(21–124), it is essentially fully as capable as the Leu-9 and RNAase-S-(1–20) peptides in directing the re-formation of correct disulfide-containing conformation of RNAase-S-(21–124) after disulfide randomization of the latter.