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 1H and 13C peptide chemical shift differences were analysed and a classification of IL anions according to the Hofmeister series was derived. The observed chemical shift changes indicate significant interactions between the peptide and the ILs. In addition, we examined the impact of different ILs towards the cis/trans equilibrium state of the Xaa-Pro peptide bonds. In this context, the IL cations appear to be of exceptional importance for inducing an alteration of the native cis/trans equilibrium state of Xaa-Pro bonds in favour of the trans-isomers.

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. Using circular dichroism, fluorescence, and NMR spectroscopy, we ruled out the existence of stably populated folding intermediates, whereas UV absorbance measurements hinted at stable residual structures in the unfolded state. These residual structures proved, however, to have no impact on the folding parameters. Studies by limited proteolysis, CD, and NMR spectroscopy under non-denaturing conditions suggested pronounced dynamics of the protein in the native state, which as long as unrestrained by acidic pH or bound Ca(2+) ions exert considerable influence on the unfolding transition.

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. However, the field has received more attention in the last few years. This Review features a meta-analysis of the available data and findings and should, therefore, provide a basis for a scientifically profound understanding of the nature and mechanisms of interactions between ILs and structured or nonstructured peptides. Fundamental aspects of the interactions between different peptides/oligopeptides and ILs are complemented by sections on the experimental (spectroscopy, structural biology) and theoretical (computational chemistry) possibilities to explain the phenomena reported so far in the literature. In effect, this should lead to the development of novel applications and support the understanding of IL-solute interactions in general.

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. The formation of this type of compounds from phosphatidylcholine was compared for a series of N- and C2-substituted ethanolamine derivatives as well as non-charged alcohols such as glycerol and ethylene glycol. The rapid exchange of the choline head group by ethanolamine derivatives having a low molecular volume (diethanolamine and serinol) gave rise to an efficient production of the corresponding CL analogs. In contrast, the yields were comparably low in the reaction with bulky nitrogenous acceptor alcohols (triethanolamine, tris(hydroxymethyl)aminomethane, tetrakis(hydroxyethyl)ammonium) or the non-charged alcohols. Therefore, a strong dependence of the conversion of the monophosphatidyl to the diphosphatidyl compound on steric parameters and the head group charge was concluded. The enzyme-assisted strategy was used for the preparation of purified diphosphatidyldiethanolamine and diphosphatidylserinol.

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. However, little is known about the folding of homologous RNases. Here, we analyze the folding pathway of onconase, a homologous protein from the Northern leopard frog with great potential as a tumor therapeutic, by high-resolution techniques. Although onconase and RNase A significantly differ in the primary structure (28% sequence identity) and in thermodynamic stability (DeltaDeltaG = 20 kJ mol(-1)), both enzymes possess very similar tertiary structures. The present folding studies on onconase by rapid mixing techniques in combination with fluorescence and NMR spectroscopy allow the structural assignment of the three kinetic phases observed in stopped-flow fluorescence spectroscopy. After a slow peptidyl-prolyl cis-to-trans isomerization reaction in the unfolded state, ONC folds via an on-pathway intermediate to the native state. By quenched-flow hydrogen/deuterium exchange experiments coupled with 2D NMR spectroscopy, 31 amino acid residues were identified to be involved in the structure formation of the intermediate. Twelve of these residues are identical in the RNase A sequence, which is a significantly higher percentage (39%) than the overall 28% sequence identity. Moreover, the structure of this intermediate closely resembles two of the intermediates that occur early during the refolding of RNase A. Obviously, in spite of considerable differences in their amino acid sequence the initial folding events of both proteins are comparable, guided by a limited number of conserved residues.

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. Examinations on numerous peptides carrying the N-terminal Xaa-Xaa-Pro motif of Tat revealed that tryptophan at position two strongly enhanced DP IV inhibition and immunosuppression. Here, we present evidence that the thromboxane A2 receptor exposing N-terminal Met-Trp-Pro at the cell surface could be a potential endogenous, inhibitory DP IV ligand. Moreover, our data suggest that the major envelope proteins p37k of the orthopoxviruses variola virus and vaccinia virus, as well as the B2L antigen of the parapoxvirus orf, that also carry N-terminal Met-Trp-Pro, could mediate immunosuppressive effects. Further examinations are in progress to identify new physiologic, inhibitory DP IV ligands and to enlighten the mechanism underlying the DP IV-mediated effects.

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. (PLDStr) were compared with respect to product yield and purity as well as the initial rates in transphosphatidylation and competing hydrolysis. In all reactions, PLDStr showed a remarkably higher transphosphatidylation activity than PLDcab. However, higher yields of the phospholipids with diethanolamine, triethanolamine, and serinol were obtained by PLDcab because PLDStr resulted in the additional formation of diphosphatidyl derivatives. In the synthesis of the Tris and BisTris derivatives, PLD(Str) was much more appropriate because voluminous head group alcohols (>129A3) are poorly converted by PLDcab. With BisTris as acceptor alcohol two regioisomeric forms of phosphatidyl-BisTris were obtained.

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. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.

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). Bip-Pro inhibited the [(14)C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity (K(i) = 24 microm and 3.4 microm, respectively) in a competitive manner. By employing the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT1 or PEPT2 it was found that Bip-Pro was transported by both peptide transporters although to a much lower extent than the reference substrate, Gly-Gln. Bip-Pro remained intact to > 98% for at least 8 h when incubated with intact cell monolayers. Bip-[(3)H]Pro uptake into SKPT cells was linear for up to 30 min and pH dependent with a maximum at extracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeled Bip-Pro but also by known peptide transporter substrates such as dipeptides, cefadroxil, Ala-4-nitroanilide and delta-aminolevulinic acid, but not by glycine. Bip-Pro uptake in SKPT cells was saturable with a Michaelis-Menten constant (K(t)) of 7.6 microm and a maximal velocity (V(max)) of 1.1 nmol x 30 min(-1) x mg of protein(-1). Hence, the uptake of Bip-Pro by PEPT2 is a high-affinity, low-capacity process in comparison to the uptake of Gly-Sar. We conclude that Bip-[(3)H]Pro is a valuable substrate for both mechanistic and structural studies of H(+)/peptide transporter proteins.

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.m.). Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability is receiving much attention in pharmaceutical research. Cefuroxime exhibiting significant advantages in the parental treatment of common infections, was used as model drug in the present study. The effect of the cationic absorption enhancers (four quaternary ammonium salts) on the lipophilicity of cefuroxime was investigated by means of the n-octanol/water system. The results on partitioning coefficients in the n-octanol/buffer system were confirmed using an in vitro transport model with artificial (dodecanol collodium membrane) and biological membranes (Charles-River guinea pig).

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. Recently, we characterized the thromboxane A2 receptor peptide TXA2-R(1-9), bearing the N-terminal MWP sequence motif, as a potent DP IV inhibitor possibly playing a functional role during antigen presentation by inhibiting T cell-expressed DP IV [Wrenger, S., Faust, J., Mrestani-Klaus, C., Fengler, A., Stöckel-Maschek, A., Lorey, S., Kähne, T., Brandt, W., Neubert, K., Ansorge, S. & Reinhold, D. (2000) J. Biol. Chem.275, 22180-22186]. Here, we demonstrate that amino acid substitutions at different positions of Tat(1-9) can result in a change of the inhibition type. Certain Tat(1-9)-related peptides are found to be competitive, and others linear mixed-type or parabolic mixed-type inhibitors indicating different inhibitor binding sites on DP IV, at the active site and out of the active site. The parabolic mixed-type mechanism, attributed to both non-mutually exclusive inhibitor binding sites of the enzyme, is described in detail. From the kinetic investigations and molecular modeling experiments, possible interactions of the oligopeptides with specified amino acids of DP IV are suggested. These findings give new insights for the development of more potent and specific peptide-based DP IV inhibitors. Such inhibitors could be useful for the treatment of autoimmune and inflammatory diseases.

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. Rhodamine 110 (R110), a highly fluorescent xanthene dye, was used to synthesize dipeptidyl peptidase IV (DP IV/CD26) substrates Gly(Ala)-Pro-R110-R, thus facilitating a stable binding of the fluorescent moiety on the cell surface. The fixation resulted from the interaction with the reactive anchor rhodamine and allowed the quantification of cellular DP IV activity on single cells. The reactivity, length, and hydrophobicity of rhodamine was characterized as the decisive factor that facilitated the determination of cellular DP IV activity. Using fluorescence microscopy, it was possible to differentiate between different DP IV activities. The hydrolysis of cell-bound substrates Xaa-Pro-R110-R by DP IV of neighboring cells and by soluble DP IV was shown using flow cytometry. These data demonstrate that ectopeptidases such as DP IV may be involved in communication between blood cells via proteolysis of cell-associated substrates.