NMR spectroscopic studies of a TAT-derived model peptide in imidazolium-based ILs: influence on chemical shifts and the cis/trans equilibrium state.

NMR spectroscopy was used to study systematically the impact of imidazolium-based ionic liquid (IL) solutions on a TAT-derived model peptide containing Xaa-Pro peptide bonds. The selected IL anions cover a wide range of the Hofmeister series of ions. Based on highly resolved one- and two-dimensional NMR spectra individual H and C peptide chemical shift differences were analysed and a classification of IL anions according to the Hofmeister series was derived.

Native state dynamics affects the folding transition of porcine pancreatic phospholipase A2.

Porcine pancreatic phospholipase A2, a small and disulfide rich protein, is extremely resistant against chemically or thermally induced unfolding. Despite this marked resistance, the protein displays broad unfolding transitions resulting in comparatively low apparent thermodynamic stability. Broad unfolding transitions may result from undetected folding intermediates, residual structures in the unfolded state or an inhomogeneity of the native state.

On the nature of interactions between ionic liquids and small amino-acid-based biomolecules.

During the last decade, ionic liquids (ILs) have revealed promising properties and applications in many research fields, including biotechnology and biological sciences. The focus of this contribution is to give a critical review of the phenomena observed and current knowledge of the interactions occurring on a molecular basis. As opposed to the huge advances made in understanding the properties of proteins in ILs, complementary investigations dealing with interactions between ILs and peptides or oligopeptides are underrepresented and are mostly only of phenomenological nature.

New cardiolipin analogs synthesized by phospholipase D-catalyzed transphosphatidylation.

Cardiolipin (CL) and related diphosphatidyl lipids are hardly accessible because of the complexity of their chemical synthesis. In the present paper, the transphosphatidylation reaction catalyzed by phospholipase D (PLD) from Streptomyces sp. has been proven as an alternative enzyme-assisted strategy for the synthesis of new CL analogs.

Structural studies on ionic liquid/water/peptide systems by HR-MAS NMR spectroscopy.

The present work reports on an assessment of high-resolution magic angle spinning (HR-MAS) NMR spectroscopy for structural investigations of peptides dissolved in aqueous ionic liquids. Highly resolved one- and two-dimensional NMR spectra are obtained that allow for complete proton resonance assignments of both the peptides as solutes and the ionic liquids as solvents. Successful application of the HR-MAS method facilitates for the first time high-resolution NMR analysis of complex ionic liquid/peptide systems at the molecular level, mainly on the basis of chemical-shift changes.

The folding pathway of onconase is directed by a conserved intermediate.

A promising approach to unravel the relationship between sequence information, tertiary structure, and folding mechanism of proteins is the analysis of the folding behavior of proteins with low sequence identity but comparable tertiary structures. Ribonuclease A (RNase A) and its homologues, forming the RNase A superfamily, provide an excellent model system for respective studies. RNase A has been used extensively as a model protein for folding studies.

Non-substrate peptides influencing dipeptidyl peptidase IV/CD26 activity and immune cell function.

Investigations using inhibitors of dipeptidyl peptidase IV (DP IV) activities and DP IV-/- mice indicated an immunoregulatory role of DP IV that could not be compensated by DP IV-like enzymes. The HIV-1 Tat protein was identified as the first natural inhibitor of DP IV and as immunosuppressor. This review summarizes our investigations on the identification of the amino acid motif of Tat responsible for DP IV inhibition and of endogenous DP IV-inhibitory ligands that suppress immune cell activation.

Phospholipase D-catalyzed synthesis of new phospholipids with polar head groups.

A series of new phospholipids with polar head groups have been synthesized by enzymatic transphosphatidylation of 1,2-dioleoyl-sn-glycerophosphocholine and identified by 1H NMR and MALDI-TOF-MS. The acceptor alcohols were N- or C2-substituted derivatives of ethanolamine (diethanolamine, triethanolamine, serinol, Tris, BisTris). Phospholipases D from cabbage (PLDcab) and Streptomyces sp.

Amino acids allosterically regulate the thiamine diphosphate-dependent alpha-keto acid decarboxylase from Mycobacterium tuberculosis.

The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids.

Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters.

In this study we described the design, rational synthesis and functional characterization of a novel radiolabeled hydrolysis-resistant high-affinity substrate for H(+)/peptide cotransporters. L-4,4'-Biphenylalanyl-L-Proline (Bip-Pro) was synthesized according to standard procedures in peptide chemistry. The interaction of Bip-Pro with H(+)/peptide cotransporters was determined in intestinal Caco-2 cells constitutively expressing human H(+)/peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively expressing rat H(+)/peptide cotransporter 2 (PEPT2).

Improvement of lipophilicity and membrane transport of cefuroxime using in vitro models.

Most beta-lactam antibiotics cannot be absorbed orally and, therefore, must be administered intravenously (i.v.) or intramuscularly (i.

Different modes of dipeptidyl peptidase IV (CD26) inhibition by oligopeptides derived from the N-terminus of HIV-1 Tat indicate at least two inhibitor binding sites.

Dipeptidyl peptidase IV (DP IV, CD26) plays an essential role in the activation and proliferation of lymphocytes, which is shown by the immunosuppressive effects of synthetic DP IV inhibitors. Similarly, both human immunodeficiency virus-1 (HIV-1) Tat protein and the N-terminal peptide Tat(1-9) inhibit DP IV activity and T cell proliferation. Therefore, the N-terminal amino acid sequence of HIV-1 Tat is important for the inhibition of DP IV.

Transcellular proteolysis demonstrated by novel cell surface-associated substrates of dipeptidyl peptidase IV (CD26).

Proteolytic enzymes contribute to the regulation of cellular functions such as cell proliferation and death, cytokine production, and matrix remodeling. Dipeptidyl peptidase IV (DP IV) catalyzes the cleavage of several cytokines and thereby contributes to the regulation of cytokine production and the proliferation of immune cells. Here we show for the first time that cell surface-bound DP IV catalyzes the cleavage of specific substrates that are associated with the cellular surface of neighboring cells.

Thioxo amino acid pyrrolidides and thiazolidides: new inhibitors of proline specific peptidases.

Aminopeptidase P (APP), dipeptidyl peptidase II (DP II), dipeptidyl peptidase IV (DP IV) and prolyl oligopeptidase (POP) are proline specific peptidases. Hence, they are able to cleave peptide bonds containing the imino acid proline. Amino acid pyrrolidides (Pyrr) and thiazolidides (Thia) are well-known product analogue inhibitors of DP IV and POP.

Down-regulation of T cell activation following inhibition of dipeptidyl peptidase IV/CD26 by the N-terminal part of the thromboxane A2 receptor.

Using synthetic inhibitors, it has been shown that the ectopeptidase dipeptidyl peptidase IV (DP IV) (CD26) plays an important role in the activation and proliferation of T lymphocytes. The human immunodeficiency virus-1 Tat protein, as well as the N-terminal nonapeptide Tat(1-9) and other peptides containing the N-terminal sequence XXP, also inhibit DP IV and therefore T cell activation. Studying the effect of amino acid exchanges in the N-terminal three positions of the Tat(1-9) sequence, we found that tryptophan in position 2 strongly improves DP IV inhibition.

Effects of nonapeptides derived from the N-terminal structure of human immunodeficiency virus-1 (HIV-1) Tat on suppression of CD26-dependent T cell growth.

The human immunodeficiency virus-1 (HIV-1) transactivator Tat occurs extracellularly and exerts immunosuppressive effects. Interestingly, Tat inhibits dipeptidyl peptidase IV (DP IV) activity of the T cell activation marker CD26. The short N-terminal nonapeptide Tat(1-9), MDPVDPNIE, also inhibits DP IV activity and suppresses DNA synthesis of tetanus toxoid-stimulated peripheral blood mononuclear cells (PBMC).

N-terminal HIV-1 Tat nonapeptides as inhibitors of dipeptidyl peptidase IV. Conformational characterization.

Compared to the N-terminal nonapeptide of the HIV-1 Tat protein as inhibitor of activity of DP IV which is supposed to mediate the immunosuppressive effects of HIV-1 Tat, the Ile5 and Leu6 analogues showed strongly reduced inhibitory activity. Interestingly, replacement of Asp2 with Gly or Lys led to compounds with considerably enhanced inhibition. Therefore, we have applied 1H NMR spectroscopy and restrained molecular dynamics calculations to elucidate the molecular conformation of a series of Tat nonapeptides.

1H NMR conformational study on N-terminal nonapeptide sequences of HIV-1 Tat protein: a contribution to structure-activity relationships.

On the basis of our recent results, the N-terminal sequence of HIV-1 Tat protein as a natural competitive inhibitor of dipeptidyl peptidase IV (DP IV) is supposed to interact directly with the active site of DP IV hence mediating its immunosuppressive effects via specific DP IV interactions. Of special interest is the finding that amino acid substitutions of the Tat(1-9) peptide (MDPVDPNIE) in position 5 with S-isoleucine and in position 6 with S-leucine led to peptides with strongly reduced inhibitory activity suggesting differences in the solution conformation of the three analogues. Therefore, 1H NMR techniques in conjunction with molecular modelling have been used here to determine the solution structure of Tat(1-9), I5-Tat(1-9) and L6-Tat(1-9) and to examine the influence of amino acid exchanges on structural features of these peptides.

The N-terminal structure of HIV-1 Tat is required for suppression of CD26-dependent T cell growth.

Evidence exists that the human immunodeficiency virus-1 (HIV-1) transactivator Tat occurs extracellularly and is involved in the immunosuppression of non-HIV-1-infected T cells of acquired immunodeficiency syndrome (AIDS) patients. The mechanism of this immunosuppressive activity of Tat has been controversially discussed. Interestingly, Tat binds to the T cell activation marker CD26, which has been shown to play a key role in the regulation of growth of lymphocytes and to inhibit its dipeptidyl peptidase IV (DP IV) activity.

Structural modifications of the N-terminal tetrapeptide segment of [D-Ala2]deltorphin I: effects on opioid receptor affinities and activities in vitro and on antinociceptive potency.

A series of deltorphin I analogs containing D- or L-N-methylalanine (MeAla), D- or L-proline (Pro), alpha-aminoisobutyric acid (Aib), sarcosine (Sar) or D-tert-leucine (Tle) in place of D-Ala2, or phenylalanine in place of Tyr1, was synthesized. The opioid activity profiles of these peptides were determined in mu and delta opioid receptor-representative binding assays and bioassays in vitro as well as in the rat tail flick test in vivo. In comparison with the deltorphin I parent, both the L- and the D-MeAla2-analog were slightly more potent delta agonists in the mouse vas deferens (MDV) assay, and the D-MeAla2-analog showed two-fold higher antinociceptive potency in the analgesic test.

Proton NMR conformational analysis of cyclic beta-casomorphin analogues of the type Tyr-cyclo[N omega-D-Orn-Xaa-Yaa-Gly-].

A conformational study of the cyclic beta-casomorphin-5 analogues H-Tyr-cyclo[-D-Orn-2-Nal-Pro-Gly-] (1) (mu-selective agonist; 2-Nal = 2 naphthylalanine), H-Tyr-cyclo[-D-Orn-2-Nal-D-Pro-Gly-] (2) (mixed mu agonist/delta antagonist) and H-Tyr-cyclo[-D-Orn-Phe-D-Pro-Gly-] (3) (highly potent mu and delta agonist) has been carried out using 1H NMR spectroscopy. A complete assignment of the proton resonances of the three pentapeptides has been achieved. Compound 1 was shown to exist in two conformations, a major one (90%) characterized by a cis amide bond between 2-Nal3 and Pro4, and a minor one (10%) showing cis amide bonds both between D-Orn2 and 2-Nal3 and between 2-Nal3 and Pro4.

Synthesis of dipeptide 4-nitroanilides containing non-proteinogenic amino acids.

A series of tert-butyloxycarbonyl amino acid 4-nitroanilides, including N-alkylated amino acids and (R)-thiazolidine-4-carboxylic acid, (S)-oxazolidine-4-carboxylic acid. (4S,5S)-5-methyloxazolidine-4-carboxylic acid, (4S,5R)-5-methyloxazolidine-4-carboxylic acid, (S)-azetidine-2-carboxylic acid, (S)-pipecolic acid and (S)-3,4-dehydroproline, were prepared conveniently by the isocyanate method or the mixed anhydride procedure. The resulting amino acid 4-nitroanilides were extended to corresponding dipeptide 4-nitroanilides with tert-butyloxycarbonyl-(S)-alanine.

Cyclic beta-casomorphin analogues with mixed mu agonist/delta antagonist properties: synthesis, pharmacological characterization, and conformational aspects.

Analogues of the potent and moderately mu-opioid-receptor-selective cyclic beta-casomorphin-5 derivative H-Tyr-c[-D-Orn-Phe-D-Pro-Gly-] (2) were prepared by conventional solution synthesis. Replacement of the Phe3 residue by 2-naphthylalanine (2-Nal) led to a peptide (4) with high affinity for both mu and delta opioid receptors. This compound turned out to be an agonist in the mu-receptor-representative guinea pig ileum (GPI) assay but a moderately potent antagonist against various delta agonists in the delta-receptor-representative mouse vas deferens (MVD) assay.