Binding Of Dipeptides Research Articles

Enantioselective binding of dipeptides using acyclic receptors

Novel receptors featuring a 2,6-diamidopyridine ‘head’ group and bearing sulfonamidopeptide sidearms have been prepared on the solid-phase; one receptor showed high selectivity for N-Cbz-D-Ala-D-AlaOH over its enantiomer N-Cbz-L-Ala-L-AlaOH, but absolute binding constants were relatively weak, which can be understood in terms of receptors which have to unfold, breaking intramolecular hydrogen bonds, in order to accommodate the guests.

Dipeptide binding to the extended active site of the Streptomyces R61 D-alanyl-D-alanine-peptidase: the path to a specific substrate.

Bacterial cell walls are cross-linked in the final step of biosynthesis by specific D-alanyl-D-alanine(DD)-peptidases/transpeptidases. The natural substrates of these enzymes should therefore be segments of peptidoglycan, but high specificity for such structures has yet to be demonstrated. The binding of dipeptides to the extended substrate binding site of the Streptomyces R61 DD-peptidase has been studied by means of a fluorescent beta-lactam probe. It was found that dipeptides of structure Gly-L-Xaa have affinity for a subsite adjacent to the beta-lactam binding site. Hydrophobic peptides such as Gly-L-Met and Gly-L-aminocaprylic acid had the greatest affinity for this site, with dissociation constants in each case of 0.19 mM. A combination of this motif with the C-terminal D-alanyl-D-alanine moiety required of a DD-peptidase substrate yielded a new substrate, glycyl-L-alpha-amino-epsilon-pimelyl-D-alanyl-D-alanine. Steady-state kinetic measurements established this compound as the most specific peptide substrate yet discovered for a DD-peptidase by at least 3 orders of magnitude (k(cat) = 69 s(-1), K(m) = 7.9 microM, k(cat)/K(m) = 8.7 x 10(6) s(-1) M(-1)); acylation was rate-determining at saturation. This substrate, presumably not coincidentally, contains the acyl donor and acceptor moieties, appropriately separated, of the Streptomyces peptidoglycan structure. This general method of approach should be of value in the search for specific substrates and inhibitors (antibiotics) of other DD-peptidases.

Interference of rheumatoid factor activity by aspartame, a dipeptide methyl ester.

Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L-Asp-L-Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo- and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. (1998). Clin. Pharmac. Ther. 63, 580-593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid-phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl(2) to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp-Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG(1) antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp-Phe and Phe-Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer-assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG(4) antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints.

Interference of rheumatoid factor activity by aspartame, a dipeptide methyl ester

Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L-Asp-L-Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo- and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. (1998). Clin. Pharmac. Ther. 63, 580–593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid-phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl2 to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp–Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG1 antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp–Phe and Phe–Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer-assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG4 antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints. Copyright © 1999 John Wiley & Sons, Ltd.

Multisite Recognition of Aqueous Dipeptides by Oligoglycine Arrays Mixed with Guanidinium and Other Receptor Units at the Air−Water Interface

Equimolar mixed monolayers of dioctadecyl glycylglycinamide amphiphile (2C 18 BGly 2 NH 2 ) with other functional amphiphiles bearing guanidinium, pyridine, and alcoholic OH groups were prepared on water, and the binding of aqueous dipeptides to these monolayers was investigated by π-A isotherm measurement, FT-IR spectroscopy, and XPS elemental analysis. The binding behavior of GlyLeu to the mixed monolayer of 2C 18 BGly 2 NH 2 and guanidinium amphiphile (2C 18 BGua) was analyzed by a Langmuir isotherm to give a saturation guest/amphiphile ratio (a) of 0.46 and a binding constant (K) of 6400 M -1 . The former value indicates that the binding site for one GlyLeu molecule was formed cooperatively by the two monolayer components. The binding constant is much enhanced relative to those observed for the 2C 18 BGly 2 NH 2 single-component monolayer (35 M -1 ) and an equimolar mixed monolayer of 2C 18 BGly 2 NH 2 and benzoic acid amphiphile (2C 18 BCOOH) (475 M -1 ). When the second amphiphile was replaced with a pyridine amphiphile (2C 18 Py) or with an alcohol amphiphile (2C 18 OH), binding constants for GlyLeu were lowered to 124 and 43 M -1 , respectively. The enhanced binding in the former is attributed to strong guanidinium-carboxylate interaction upon C-terminal guest insertion and stable antiparallel hydrogen bonding among peptide chains. The binding of a second dipeptide, LeuGly, to the mixed monolayer of 2C 18 BGly 2 NH 2 / 2C 18 BGua gave a K value (2170 M -1 ) that is only one-third of that of GlyLeu. The difference is apparently related to the disposition of the hydrophobic side chain of the Leu residue in the C-terminal insertion. Thus, size matching of side chains of amino acid residues in host and guest determines selectivity of binding. Guest dipeptides are bound to the host most efficiently when the separation of host peptide chains is suited for the formation of strong hydrogen bonds between host and guest.

X-ray crystal structure of a dipeptide-chymotrypsin complex in an inhibitory interaction.

The dipeptide D-leucyl-L-phenylalanyl p-fluorobenzylamide (D-Leu-Phe-NH-BzlF) inhibits chymotrypsin strongly in a competitive manner with the Ki value of 0.61 microM [Shimohigashi, Y., Maeda, I., Nose, T., Ikesue, K., Sakamoto, H., Ogawa, T., Ide, Y., Kawahara, M., Nezu, T., Terada, Y., Kawano, K. & Ohno, M. (1996) J. Chem. Soc. Perkin Trans. 1, 2479-2485]. The structure/activity studies have suggested a unique inhibitory conformation, in which the C-terminal benzyl group fits the chymotrypsin S1 site and the hydrophobic core constructed by the side chains of D-Leu-Phe fits the S2 or S1' site. To verify this assumption, the molecular structure of the complex between the dipeptide and gamma-chymotrypsin has been determined crystallographically. Gamma-chymotrypsin itself was first crystallized and refined at 1.6-A resolution. The refined structure was virtually identical to the conformation reported and the electron density at the active site was interpreted as a pentapeptide Thr-Pro-Gly-Val-Tyr derived from autolysis of the enzyme (residues 224-228). The chymotrypsin-dipeptide complex was obtained by soaking the crystals of gamma-chymotrypsin in a solution saturated with the dipeptide inhibitor. The crystal structure of the complex has been refined at 1.8-A resolution to a crystallographic R-factor of 18.1%. The structure of gamma-chymotrypsin in the complex agreed fairly well with that of gamma-chymotrypsin per se with a rmsd of 0.13 A for all the C alpha carbons. Two inhibitor molecules were assigned in an asymmetric unit, i.e. one in the active site and the other at the interface of two symmetry-related enzyme molecules. In both sites dipeptides adopted very similar folded conformations, in which side chains of D-Leu-Phe are spatially proximal. In the active site where the binding of dipeptide was judged to be a direct cause of inhibition, C-terminal p-fluorobenzylamide group of the dipeptide, NH-BzlF, was found in the S1 hydrophobic pocket. At the bottom of this pocket, the p-fluorine atom hydrogen bonded with a water molecule, probably to enhance the inhibitory activity. The stereospecific interaction of R and S isomers of the dipeptide with C-terminal NH-C*H(CH3)-C6H5 was well explained by the space available for methyl replacement in the complex. The hydrophobic core constructed by side chains of D-Leu-Phe was found at the broad S2 site. Interestingly, a novel interaction was found between the inhibitor Phe residue and chymotrypsin His57, the phenyl of Phe and the imidazole of His being in a pi-pi stacking interaction at a distance 3.75 A.

Multi-Site Binding of Aqueous Dipeptides by Mixed Monolayers at the Air-Water Interface

Abstract Aqueous dipeptides are bound to two-component monolayers which contain oligopeptide polar units together with benzoic acid or pyridine head groups. Binding of guest dipeptides to the mixed monolayers depends specifically on the nature of interfacial cavities which can be controlled by combination of polar head groups. The binding ratio is lessened with increasing sizes of the side chain of the Y residue in the Gly-Y dipeptide moiety of the host amphiphiles.

Molecular Recognition of Aqueous Dipeptides at Multiple Hydrogen-Bonding Sites of Mixed Peptide Monolayers

Oligopeptide amphiphiles with different dipeptide moieties of −XYNH2 (X = Gly and Ala, Y = Gly, Ala, Val, Leu, and Phe) were synthesized. Binding of aqueous dipeptides onto monolayers of equimolar mixtures of these amphiphiles with a benzoic acid amphiphile (2C18BCOOH) was investigated by π−A isotherm measurement, FT-IR spectroscopy, and XPS elemental analysis. For given GlyX dipeptides (X = neutral and hydrophobic residues), the binding ratio was lessened with increasing sizes of the side chain of the Y residue in the GlyY dipeptide moiety of the host amphiphiles. The Langmuir-type saturation behavior was observed for binding of GlyLeu to an equimolar monolayer of 2C18BGly2NH2 and 2C18BCOOH. Its binding constant of 475 M-1 was 10 times larger than that observed for a single-component monolayer of 2C18BGly2NH2 (K = 35 M-1). The saturation guest/host ratio was 0.47. The mode of substrate insertion into the binding site was examined by FT-IR spectroscopy. When the hydrophobic residue was on the C-terminal of a guest dipeptide (GlyX), the C-terminal insertion was selected with accompanying formation of cyclic carboxylic acid dimers at the interface. In the case of XGly guests, the N-terminal insertion with salt bridge formation with the host was observed. When the two residues of a dipeptide had close hydrophobicities, both C- and N-terminal insertions were observed. Formation of these binding sites is apparently induced by dipeptide binding.

Molecular Recognition of Aqueous Dipeptides by Noncovalently Aligned Oligoglycine Units at the Air/Water Interface

Abstract: Binding of aqueous dipeptides, GlyX and X'Gly (X = Leu, Phe, Pro, and Ala; X' = Leu and Phe), onto monolayers of dialkyl oligoglycyl amphiphiles has been investigated by n-A isotherm measurement, FT-IR spectroscopy, and XPS elemental analysis. Dipeptides with the N-terminal glycine residue (GlyX) were selectively bound onto monolayers of an amphiphile in which dioctadecylamine moiety was connected with the glycylglycinamide head group via the terephthaloyl unit. When the dipeptide (GlyLeu) concentration in the subphase was varied, the Langmuir-type saturation behavior was observed with the equimolar binding and the binding constant of 35 M-I. The binding efficiency increased in the order of GlyPhe > GlyLeu > GlyPro > GlyAla, implying that the binding is promoted by hydrophobic interaction. The binding was not detected when either of the terephthaloyl and glycylglycinamide units were absent in the monolayer component. Dipeptides with the C-terminal glycine residue (X'Gly) were not bound at all. These results are satisfied by a molecular model in which guest peptides were inserted into the monolayer from the C-terminal. The observed binding selectivity is explained by hydrophobic interaction between the side chain of the C-terminal residue and the hydrophobic cavity in the monolayer and formation of stable antiparallel hydrogen bonding between guest dipeptides and host diglycine chains. The formation of a specific binding site by noncovalent self-assembly leads to a new thinking in the monolayer research.

Explanation for the non-specificity of small peptide-antibody interaction

Testing the reactivity of synthetic peptides with monoclonal antibodies provides information on protein-protein interactions. Starting from a minimal basis, that is considering dipeptide-antibody interactions, binding occurs in a reproducible manner. With no knowledge of the testing antibody specificity, peptide length is the only parameter which can be easily handled. Best binding dipeptides are then extended by adding one aminoacid to either end, and the process goes on with best binding tri, tetra, etc., -peptides. Such a process would be well founded if specificity was gained at each step. In fact, a computer analysis of experimental results shows that binding of dipeptide tends to be independent from the specificity of the antibody. An ad hoc method mainly based on ranking and sorting operations, assesses predominant factor(s) accounting for dipeptide reactivity. Moreover, properties of dipeptides are not generalisable to tripeptides. The continuity of the extension process cannot be assumed.

Interaction between Squash Inhibitors and Bovine Trypsinogen

Squash seeds proteinase inhibitors form stoichiometric complexes with bovine trypsinogen. In terms of association constants (Ka), the interaction is weak. The inhibitors bind to the zymogen with Ka values of approx. 10(4)M-1 i.e. 2 X 10(7) times weaker than to bovine beta-trypsin. Squash inhibitor with Lys at the P1 position binds to trypsinogen with a Ka value 2.1-fold higher than the inhibitor with Arg at P1. The Ile-Val binding cleft and the Ca2+ binding site of trypsinogen are cooperatively linked to the inhibitor binding site. Although these three sites are spatially separated, either binding of calcium ion or Ile-Val dipeptide to trypsinogen increase the Ka values 3-fold and more than 100-fold, respectively. In the presence of Ile-Val trypsinogen resynthetizes extremely slowly (about 10(4) times slower than beta-trypsin) the reactive site peptide bond in squash inhibitors.

An empirical potential for interactions of large molecules: Application to binding of dipeptides to DNA

AbstractAn empirical potential is introduced that is suitable for calculation of the interaction enegies of biomolecules with thousands of atoms. The potential consists of electrostatic, repulsion, and dispersion energy terms. The approach used for parametrization of the potential is entirely different from that used with other existing potentials. Namely, all the terms were parametrized independently to retain their physical significance. The sum of the electrostatic and repulsion terms mimic the SCF interaction energy calculated using Huzinaga's minimal basis set MINI‐1. The dispersion energy is very important and is usually predominant for the interactions of large (polar) molecules in the gas phase as well as in the liquid phase.

The binding of a spin-labelled dipeptide to papain

The binding of a spin-labelled dipeptide to papain