Dipeptide Ligands Research Articles

Broad-Spectrum Clearance of Lipopolysaccharides from Blood Based on a Hemocompatible Dihistidine Polymer.

Blood infection can release toxic bacterial lipopolysaccharides (LPSs) into bloodstream, trigger a series of inflammatory reactions, and eventually lead to multiple organ dysfunction, irreversible shock, and even death, which seriously threatens human life and health. Herein, a functional block copolymer with excellent hemocompatibility is proposed to enable broad-spectrum clearance of LPSs from whole blood blindly before pathogen identification, facilitating timely rescue from sepsis. A dipeptide ligand of histidine-histidine (HH) was designed as the LPS binding unit, and poly[(trimethylamine N-oxide)-co-(histidine-histidine)], a functional block copolymer combining the LPS ligand of HH and a zwitterionic antifouling unit of trimethylamine N-oxide (TMAO), was then designed by reversible addition-fragmentation chain transfer (RAFT) polymerization. The functional polymer achieved effective clearance of LPSs from solutions and whole blood in a broad-spectrum manner and had good antifouling and anti-interference properties and hemocompatibility. The proposed functional dihistidine polymer provides a novel strategy for achieving broad-spectrum clearance of LPSs, with potential applications in clinical blood purification.

Dipeptide-Capped Copper Nanoparticles as Chiral Nanozymes for Colorimetric Enantioselective Recognition of 3,4-Dihydroxy-d,l-phenylalanine.

In pharmaceutical and biomedical applications, it is imperative to identify chiral molecules. However, colorimetric sensing enantiomers relying on chiral nanozymes is still a major challenge in chirality recognition. Herein, we report a facile and simple strategy to prepare copper nanoparticles (CuNPs) using d-cysteine-d-histidine (DCDH), d-cysteine-l-histidine, and l-cysteine-d-histidine as the capping agents. All of these CuNPs exhibited peroxidase-mimicking activity in 3,3',5,5'-tetramethylbenzidine oxidation and presented chiral selectivity toward 3,4-dihydroxy-d,l-phenylalanine (d,l-DOPA). More importantly, DCDH-modified CuNPs (DCDH@CuNPs) showed higher peroxidase-mimicking catalytic activity in the presence of d-DOPA than l-DOPA. This demonstrates that in the stereoselective recognition CuNPs play the catalytic center role and chiral dipeptide ligands play the inducer role. The insights obtained from this study not only provide information to deeply understand the molecular principles of colorimetric chiral recognition upon CuNPs but also guide the design of dipeptide-based chiral nanozymes toward enantiomers.

Dipeptide Ligands of TSPO

Dipeptide Ligands of TSPO

Isophthaloylbis (Azanediyl) Dipeptide Ligand and Its Complexes: Structural Study, Spectroscopic, Molecular Orbital, Molecular Docking, and Biological Activity Properties

The ligand 2,2′-(isophthaloylbis(azanediyl))bis(3-phenylpropanoic acid; H2L) was used to build novel metal complexes of Co(II), Ni(II), Fe(III), and Zn(II) metal ions. Elemental analyses, thermal, infrared, mass, electronic spectra, magnetic moments, X-ray diffraction analysis, and conductivity measurements were used to predict the nature of bonding and the stereochemistry of the complexes. The complexes were proposed to have the general formula [M(H2L)(H2O)2]Cl n , where M = Co(II), Ni(II), and Zn(II) (n = 2) and Fe(III) (n = 3), based on the elemental analyses data. All of the complexes were electrolytes, according to the molar conductivity measurements. The FT-IR spectra revealed that the dipeptide ligand is tetradentately coordinated to metal ions, with NNOO donor atoms participating in coordination with metal (II)/(III) ions, resulting in octahedral complexes. Using the recommended programme, the structural formula for the investigated ligand was optimized. Using the density-functional theory (DFT) approach, the energy gaps and other essential theoretical parameters were calculated. The agar diffusion method was used to test the in vitro biological characteristics of the ligand (H2L) and its transition metal complexes against Gram(+) bacteria (Bacillis subtilis and Staphylococcus aureus) and Gram(−) bacteria (Escherichia coli and Pseudomonas aeruginosa). The title compounds were found to be more biologically active than the parent dipeptide ligand. The title compounds have a good interaction with the crystal structures of 3t88-Escherichia coli, 3ty7-S. aureus, 5h67-Bacillus subtilis, and 5i39-Pseudomonasaeruginosa, according to molecular docking study.

Enantiomeric Separation and Molecular Modelling of Bioactive 4-Aryl-3,4-dihydropyrimidin-2(1H)-one Ester Derivatives on Teicoplanin-Based Chiral Stationary Phase

The enantiomeric separation of 15 racemic 4-aryl-3,4-dihydropyrimidin-2(1H)-one (DHP) alkoxycarbonyl esters, some of which proved to be highly active as A2B adenosine receptor antagonists, was carried out by HPLC on ChirobioticTM TAG, a chiral stationary phase (CSP) bearing teicoplanin aglycone (TAG) as the chiral selector. The racemic compounds were separated under polar organic (PO) conditions. Preliminarily, the same selectands were investigated on three different Pirkle-type CSPs in normal-phase (NP) conditions. A baseline separation was successfully obtained on TAG-based CSPs for the majority of compounds, some of which achieved high enantioselectivity ratios (α > 2) in contrast with the smaller α values (1–1.5) and the lack of baseline resolution observed with the Pirkle-type CSPs. In particular, the racemic tetrazole-fused DHP ester derivatives, namely compounds 8 and 9, were separated on TAG-based HPLC columns with noteworthy α values (8.8 and 6.0, respectively), demonstrating the potential of the method for preparative purposes. A competition experiment, carried out with a racemic analyte (6) by adding N-acetyl-d-alanine (NADA) to the mobile phase, suggested that H-bonding interactions involved in the recognition of the natural dipeptide ligand d-Ala-d-Ala into the TAG cleft should be critical for enantioselective recognition of 4-aryl DHPs by TAG. The X-ray crystal structure of TAG was elucidated at a 0.77 Å resolution, whereas the calculation of molecular descriptors of size, polar, and H-bond interactions, were complemented with molecular docking and molecular dynamics calculations, shedding light on repulsive (steric effects) and attractive (H-bond—polar and apolar) interactions between 4-aryl DHP selectands and TAG chiral selectors.

Designed leucine-rich repeat proteins bind two muramyl dipeptide ligands.

Designed protein receptors hold diagnostic and therapeutic promise. We now report the design of five consensus leucine-rich repeat proteins (CLRR4-8) based on the LRR domain of nucleotide-binding oligomerization domain (NOD)-like receptors involved in the innate immune system. The CLRRs bind muramyl dipeptide (MDP), a bacterial cell wall component, with micromolar affinity. The overall Kd app values ranged from 1.0 to 57 μM as measured by fluorescence quenching experiments. Biphasic fluorescence quenching curves were observed in all CLRRs, with higher affinity Kd1 values ranging from 0.04 to 4.5 μM, and lower affinity Kd2 values ranging from 3.1 to 227 μM. These biphasic binding curves, along with the docking studies of MDP binding to CLRR4, suggest that at least two MDPs bind to each protein. Previously, only single MDP binding was reported. This high-capacity binding of MDP promises small, soluble, stable CLRR scaffolds as candidates for the future design of pathogen biosensors.

Synthesis and Structure—Activity (Anxiolytic) Relationship Analysis of Leucyltryptophan Ligands of 18-KDA Translocator Protein

Previously, researchers at Zakusov State Institute of Pharmacology used an original strategy for designing pharmacologically active dipeptides based on non-peptide drug structures to prepare the dipeptide ligands of 18-kDa translocator protein (TSPO) N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23) and N-phenylpropionyl-L-tryptophanyl-L-leucine amide (GD-102) that showed anxiolytic activity in standard behavioral tests. The present work reports GD-102 analogs with the reverse amino-acid sequence, i.e., N-phenylpropionyl-L-leucyl-L-tryptophan methylamide (GD-140), N-phenylpropionyl-L-leucyl-L-tryptophan (GD-139), N-phenylacetyl-L-leucyl-L-tryptophan amide (GD-141), N-phenylpropionyl-L-leucyl-L-tryptophan methyl ester (GD-138), and N-phenylpropionyl-L-leucyl-L-tryptophan amide (GD-136). Open-field (OF) tests of mice after i.p. administration showed that the newly synthesized dipeptides were less active than GD-102 (0.01 – 0.1 mg/kg). Dipeptide GD-140 showed anxiolytic activity at doses of 0.1, 0.5, and 1 mg/kg; GD-139, 0.5 and 5.0; GD-141, 1 and 5. Dipeptide GD-138 at a dose of 0.1 mg/kg reduced locomotor activity of animals in the OF test. GD-136 was inactive in the dose range 0.1 – 5 mg/kg. Molecular docking studies demonstrated that the studied compounds could be placed at the TSPO binding site of ligand PK 11195 (PDB ID: 2MGY). Also, π-stacking of the phenyl groups of dipeptides GD-136, GD-139, GD-140, and GD-141 with Trp107 of the receptor was revealed. For dipeptides GD-140 and GD-102, π-stacking with Trp95 was also detected. GD-138 did not exhibit π-stacking with either Trp107 or Trp95. It was concluded that the key interaction affecting the manifestation of anxiolytic activity was π-stacking of the dipeptide ligand with Trp95 of the receptor. The Trp-Leu sequence was preferred over the Leu-Trp sequence for the TSPO dipeptide ligands.

Синтез и анализ зависимости анксиолитического эффекта от структуры лейцилтриптофановых лигандов 18 кДа транслокаторного белка

Ранее в НИИ фармакологии имени В. В. Закусова с использованием оригинальной стратегии создания фармакологически активных дипептидов на основе структур непептидных лекарственных средств получены дипептидные лиганды 18 кДа транслокаторного белка (TSPO), амид N-карбобензокси-L-триптофанил-L-изолейцина (ГД-23) и амид N-фенилпропионил-L-триптофанил-L-лейцина (ГД-102), проявившие анксиолитическую активность в стандартных тестах. В настоящей работе получены аналоги дипептида ГД-102 с обратной последовательностью аминокислотных остатков: метиламид N-фенилпропионил-L-лейцил-L-триптофана (ГД-140), N-фенилпропионил-L-лейцил-L-триптофан (ГД-139), амид N-фенилацетил-L-лейцил-L-триптофана (ГД-141), метиловый эфир N-фенилпропионил-L-лейцил-L-триптофана (ГД-138), амид N-фенилпропионил-L-лейцил-L-триптофана (ГД-136). Исследование в тесте «Открытое поле» (ОП) на мышах при внутрибрюшинном введении показало, что вновь синтезированные дипептиды уступают по активности ГД-102 (0,01 – 0,1 мг/кг). Дипептид ГД-140 проявлял анксиолитическую активность в дозах 0,1, 0,5, 1 мг/кг; ГД-139 в дозах 0,5 и 5 мг/кг; ГД-141 в дозах 1 и 5 мг/кг. Дипептид ГД-138 в дозе 0,1 мг/кг снижал двигательную активность животных в ОП. ГД-136 не проявил активность в интервале доз 0,1 – 5 мг/кг. Молекулярный докинг продемонстрировал возможность размещения изученных соединений в зоне связывания лиганда PK11195 с TSPO (PDB ID: 2MGY). При этом для ГД-136, ГД-139, ГД-140 и ГД-141 выявлено наличие pi-стэкинга фенильного фрагмента молекулы с Trp107 рецептора. У ГД-140 и ГД-102 выявлен еще и pi-стэкинг с Trp95. У ГД-138 pi-стэкинг отсутствовал как с Trp107, так и с Trp95. Сделано заключение о том, что ключевым взаимодействием, влияющим на проявление анксиолитической активности, является pi-стэкинг дипептидного лиганда с Trp95 рецептора, а последовательность Trp-Leu для дипептидных лигандов TSPO предпочтительнее последовательности Leu-Trp.

Chiral AuCuAu Heterogeneous Nanorods with Tailored Optical Activity

AbstractHere, a facile wet‐chemistry route for the selective growth of crystalline copper (Cu) along the sides of gold nanorods (Au NRs) in the presence of a hexadecylamine (HDA) is reported. The resulting heterostructures feature part etching of copper by galvanic replacement reaction and form crystalline AuCu alloy metal on one side of the Au NRs. By virtue of the dipeptide (cysteine‐phenylalanine, Cys‐Phe) ligand used during synthesis, the AuCuAu heteronanorods (HNRs) exhibit strong circular dichroism (CD) in the wavelength range of 400–1000 nm. The plasmonic chirality can be tailored by increasing the length of the Au NRs, the scale of Cu nanocrystals on the Au NRs, and the amount of gold chloride for postgrowth, resulting in an anisotropy factor (g factor) as high as 0.57 × 10−2. The strong CD signals are attributed to the local electromagnetic field. Under circular polarized light (CPL) illumination, the chiral plasmonic AuCuAu nanostructure exhibits high efficiency for light polarization dependent reactive oxygen species (1O2) that is 22.31 times that of Au NRs. The results of this study demonstrate that the chiral enantiomer provides a chirality dependent avenue for highly efficient phototherapy.

Design, Synthesis and Anxiolytic Activity Evaluation of N-Acyltryptophanyl- Containing Dipeptides, Potential TSPO Ligands.

The 18 kDa translocator protein (TSPO), previously known as the peripheral- type benzodiazepine receptor, plays a key role for the synthesis of neurosteroids by promoting transport of cholesterol from the outer to the inner mitochondrial membrane, which is the ratelimiting step in neurosteroid biosynthesis. Neurosteroids interact with nonbenzodiazepine site of GABAa receptor causing an anxiolytic effect without the side effects. Using the original peptide drug-based design strategy, the first putative dipeptide ligand of the TSPO N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23) was obtained. Molecular docking of GD-23 in the active pocket of the TSPO receptor using Glide software was carried out. The lead compounds GD-23 and its analogues were synthesized using activated succinimide esters coupling method. The anxiolytic activity of GD-23 and its analogues was investigated in vivo, using two validated behavioral tests, illuminated open field and elevated plus-maze. The in vivo studies revealed that the following parameters are necessary for the manifestation of anxiolytic activity of new compounds: the L-configuration of tryptophan, the presence of an amide group at the C-terminus, the specific size of the N-acyl substituent at the Nterminus. Compound GD-23 (N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide) demonstrated a high anxiolytic-like effect in the doses of 0.05-1.0 mg/kg i.p. comparable with that of diazepam. Compound GD-23 was also active in the open field test when was administered orally in the doses of 0.1-5.0 mg/kg. The involvement of TSPO receptor in the mechanism of anxiolytic-like activity of new compounds was proved by the antagonism of compound GD-23 with TSPO selective inhibitor PK11195 as well as with inhibitors of enzymes which are involved in the biosynthesis of neurosteroids, trilostane and finasteride. A series of N-acyl-tryptophanyl-containing dipeptides were designed and synthesized as 18 kDa translocator protein (TSPO) ligands. Using a drug-based peptide design method a series of the first dipeptide TSPO ligands have been designed and synthesized and their anxiolytic activity has been evaluated. In general, some of the compounds displayed a high level of anxiolytic efficacy comparable with that of diazepam. The involvement of TSPO receptor in the mechanism of anxiolytic activity of new compounds was proved using two methods. On this basis, the N-acyl-Ltryptophanyl- isoleucine amides could potentially be a novel class of TSPO ligands with anxiolytic activity.

The novel dipeptide ligand of TASP

Using the previously obtained first dipeptide ligand TSPO the N‑carbobenzoxy-L‑tryptophanyl-L‑isoleucine amide (GD‑23) as a basis, the new dipeptide was synthesized — the N‑phenylpropionyl–L‑tryptophanyl-L‑leucine amide (GD‑102). GD‑102 expressed anxiolytic activity in the open field test in BALB/c mice and in the elevated plus maze test in ICR mice. The minimum effective dose of GD‑102 was an order of magnitude lower than that of GD‑23. Preliminary administration of the TSPO selective antagonist, compound PK11195, completely blocked the anxiolytic activity of GD‑102, that indicated the participation of TSPO in the realization of the anxiolytic action GD‑102. The results were confirmed by molecular docking data.

A Novel Dipeptide Ligand of TSPO.

On the basis of the first dipeptide ligand of TSPO, N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23), which was obtained by us earlier, we synthesized a new dipeptide, N-phenylpropionyl-L-tryptophanyl-L-leucine amide (GD-102). GD-102 exhibited anxiolytic activity in the open field test in BALB/c mice and in the elevated plus maze test in ICR mice. The minimum effective dose of GD-102 was one order of magnitude lower than that of GD-23. Compound PK11195, a selective antagonist of TSPO, completely blocked the anxiolytic activity of GD-102, which testified to the involvement of TSPO in the realization of the anxiolytic effect of GD-102. The results were confirmed by molecular docking data.

Synthesis and characterization of Co3O4 immobilized on dipeptide-functionalized silica-coated magnetite nanoparticles as a catalyst for the selective aerobic oxidation of alcohols

A supermagnetic core–shell nanocatalyst functionalized with a dipeptide ligand as a recyclable catalyst for the selective oxidation of alcohols.

Peptide-functionalized semiconductor surfaces: strong surface electronic effects from minor alterations to backbone composition.

The use of non-canonical amino acids is a powerful way to control protein structure. Here, we show that subtle changes to backbone composition affect the ability of a dipeptide to modify solid surface electronic properties. The extreme sensitivity of the interactions to the peptide structure suggests potential applications in improving the performance of electronic devices.

Molecular Insight from DFT Computations and Kinetic Measurements into the Steric Factors Influencing Peptide Bond Hydrolysis Catalyzed by a Dimeric Zr(IV)-Substituted Keggin Type Polyoxometalate.

Peptide bond hydrolysis of several peptides with a Gly-X sequence (X = Gly, Ala, Val, Leu, Ile, Phe) catalyzed by a dimeric Zr(IV)-substituted Keggin type polyoxometalate (POM), (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]·7H2O (1), was studied by means of kinetic experiments and (1)H NMR spectroscopy. The observed rate of peptide bond hydrolysis was found to decrease with increase of the side chain bulkiness, from 4.44 × 10(-7) s(-1) for Gly-Gly to 0.81 × 10(-7) s(-1) for Gly-Ile. A thorough DFT investigation was performed to elucidate (a) the nature of the hydrolytically active species in solution, (b) the mechanism of peptide bond hydrolysis, and (c) the influence of the aliphatic residues on the rate of hydrolysis. Formation of substrate-catalyst complexes of the dimeric POM 1 was predicted as thermodynamically unlikely. Instead, the substrates prefer to bind to the monomerization product of 1, [α-PW11O39Zr(OH)(H2O)](4-) (2), which is also present in solution. In the hydrolytically active complex two dipeptide ligands are coordinated to the Zr(IV) center of 2. The first ligand is bidentate-bound through its amino nitrogen and amide oxygen atoms, while the second ligand is monodentate-bound through a carboxylic oxygen atom. The mechanism of hydrolysis involves nucleophilic attack by a solvent water molecule on the amide carbon atom of the bidentate-bound ligand. In this process the uncoordinated carboxylic group of the same ligand acts as a general base to abstract a proton from the attacking water molecule. The decrease of the hydrolysis rate with an increase in the side chain bulkiness is mostly due to the increased ligand conformational strain in the rate-limiting transition state, which elevates the reaction activation energy. The conformational strain increases first upon substitution of Hα in Gly-Gly with the aliphatic α substituent and second with the β branching of the α substituent.

Dependence of anxiolytic effects of the dipeptide TSPO ligand GD-23 on neurosteroid biosynthesis.

The elevated plus maze test showed that GD-23 (N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide), an original dipeptide ligand of TSPO, exerted anxiolytic effect when injected intraperitoneally at a dose of 0.5 mg/kg. This effect was completely blocked by the selective neurosteroid synthesis inhibitors, enzymes trilostane and finasteride. The same inhibitors do not prevent the anxiolytic effects of the benzodiazepine tranquillizer diazepam. The results of the study indicate the selective neurosteroidogenic mechanism of the anxiolytic action of GD-23.

Divalent Metal-Ion Complexes with Dipeptide Ligands Having Phe and His Side-Chain Anchors: Effects of Sequence, Metal Ion, and Anchor.

Conformational preferences have been surveyed for divalent metal cation complexes with the dipeptide ligands AlaPhe, PheAla, GlyHis, and HisGly. Density functional theory results for a full set of complexes are presented, and previous experimental infrared spectra, supplemented by a number of newly recorded spectra obtained with infrared multiple photon dissociation spectroscopy, provide experimental verification of the preferred conformations in most cases. The overall structural features of these complexes are shown, and attention is given to comparisons involving peptide sequence, nature of the metal ion, and nature of the side-chain anchor. A regular progression is observed as a function of binding strength, whereby the weakly binding metal ions (Ba(2+) to Ca(2+)) transition from carboxylate zwitterion (ZW) binding to charge-solvated (CS) binding, while the stronger binding metal ions (Ca(2+) to Mg(2+) to Ni(2+)) transition from CS binding to metal-ion-backbone binding (Iminol) by direct metal-nitrogen bonds to the deprotonated amide nitrogens. Two new sequence-dependent reversals are found between ZW and CS binding modes, such that Ba(2+) and Ca(2+) prefer ZW binding in the GlyHis case but prefer CS binding in the HisGly case. The overall binding strength for a given metal ion is not strongly dependent on the sequence, but the histidine peptides are significantly more strongly bound (by 50-100 kJ mol(-1)) than the phenylalanine peptides.

The first dipeptide ligand of translocator protein: Design and anxiolytic activity.

On the basis of the structure of Alpidem, a pyrazolopyrimidine ligand of the translocator protein (TSPO), a dipeptide TSPO ligand, N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23), was designed and synthesized using our own original peptide design strategy. This compound exhibited anxiolytic activity in BALB/cAnN mice in the "open-field" test and in outbred CD1 mice in the "elevated plus maze" test. The stereoselectivity of the anxiolytic effect of GD-23 is demonstrated. The results of this study suggest that GD-23 is a ligand of the translocator protein, and its structure can become the basis for creating anxiolytics with a fundamentally new mechanism of action.

Tri- and tetra-substituted cyclen based lanthanide(III) ion complexes as ribonuclease mimics: a study into the effect of log Ka, hydration and hydrophobicity on phosphodiester hydrolysis of the RNA-model 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP).

A series of tetra-substituted 'pseudo' dipeptide ligands of cyclen (1,4,7,10,-tetraazacyclododecane) and a tri-substituted 3'-pyridine ligand of cyclen, and the corresponding lanthanide(III) complexes were synthesised and characterised as metallo-ribonuclease mimics. All complexes were shown to promote hydrolysis of the phosphodiester bond of 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP, τ1/2 = 5.87 × 10(3) h), a well known RNA mimic. The La(III) and Eu(III) tri-substituted 3'-pyridine lanthanide(III) complexes being the most efficient in promoting such hydrolysis at pH 7.4 and at 37 °C; with τ1/2 = 1.67 h for La(III) and 1.74 h for Eu(III). The series was developed to provide the opportunity to investigate the consequences of altering the lanthanide(III) ion, coordination ability and hydrophobicity of a metallo-cavity on the rate of hydrolysis using the model phosphodiester, HPNP, at 37 °C. To further provide information on the role that the log Ka of the metal bound water plays in phosphodiester hydrolysis the protonation constants and the metal ion stability constants of both a tri and tetra-substituted 3'pyridine complex were determined. Our results highlighted several key features for the design of lanthanide(III) ribonucelase mimics; the presence of two metal bound water molecules are vital for pH dependent rate constants for Eu(III) complexes, optimal pH activity approximating physiological pH (∼7.4) may be achieved if the log Ka values for both MLOH and ML(OH)2 species occur in this region, small changes to hydrophobicity within the metallo cavity influence the rate of hydrolysis greatly and an amide adjacent to the metal ion capable of forming hydrogen bonds with the substrate is required for achieving fast hydrolysis.

Critical role of a conserved transmembrane lysine in substrate recognition by the proton-coupled oligopeptide transporter YjdL

Proton-coupled oligopeptide transporters (POTs) utilize an electrochemical proton gradient to accumulate peptides in the cytoplasm. Changing the highly conserved active-site Lys117 in the Escherichia coli POT YjdL to glutamine resulted in loss of ligand affinity as well as inability to distinguish between a dipeptide ligand and the corresponding dipeptide amide. The radically changed pHBulk profiles of Lys117Gln and Lys117Arg mutants indicate an important role of Lys117 in facilitating protonation of the transporter; a notion that is supported by the close proximity of Lys117 to the conserved ExxERFxYY POT motif previously shown to be involved in proton translocation. These results point toward a novel dual role of Lys117 in direct or indirect interaction with both proton and peptide.