The unexpected racemization and hydrogen-deuterium exchange of the hydrogen at the α-carbon of proline analogs containing the 5-azoniaspiro[4.4]nonyl-group.

Recently, we developed a novel non-fragmenting quaternary ammonium ionization tag for the mass spectrometric sensitive sequencing of peptides, based on the N-spiro proline residue (5-azoniaspiro[4.4.]nonyl-carbonyl). Herein, we present an unexpected racemization and the hydrogen-deuterium exchange (HDX) at the α-C atom of the proline derivative under basic aqueous conditions (1% water solution of triethylamine). The deuterium atom, substituted for the α-C atom, does not undergo back-exchange under acidic aqueous conditions which makes the deuterated isotopologue a promising stabile isotope-coded internal standard for quantitative analysis by mass spectrometry. The applicability of the prepared isotopologues of the quaternary ammonium salt labeled peptides for quantification experiments using the isotopic dilution method was also examined.

Hydroxypyridinones with enhanced iron chelating properties. Synthesis, characterization and in vivo tests of 5-hydroxy-2-(hydroxymethyl)pyridine-4(1H)-one.

The synthesis of 5-hydroxy-2-(hydroxymethyl)pyridin-4(1H)-one (P1) is presented, together with the evaluation of its coordination ability towards Fe(3+), studied by a combination of chemical, computational, and animal approaches. The use of complementary analytical techniques has allowed us to give evidence of the tautomeric changes of P1 as a function of pH, and to determine their influence on the coordinating ability of P1 towards Fe(3+). The pFe(3+) value 22.0 of P1-iron complexes is noticeably higher than that of deferiprone (20.6), one of the three clinical chelating agents in therapeutic use for iron overload diseases. This is due on one side to the tautomeric change to the catechol form, and on the other to the lower protonation constant of the OH group. Bio-distribution studies on mice allowed us to confirm in vivo the efficacy of P1. Furthermore the coordinating ability toward Al(3+), Cu(2+) and Zn(2+) has been studied to evaluate the possible use of P1 against a second toxic metal ion (Al(3+)), and to envisage its potential influence on the homeostatic equilibria of essential metal ions. The chelating ability of P1 toward these ions, not higher than that of the corresponding deferiprone, contributes to render P1 a more selective iron chelator.

Clustering of sulfamic acid: ESI MS and theoretical study.

Sulfamic acid has wide application in industry and has been suggested to act as an effective nucleation agent for the formation of aerosols and cloud particles. From the point of view of the role that sulfamic acid may play in aerosol formation, the study of its homoaggregation is important. Gas phase clustering study was performed for sulfamic acid H3N·SO3, (ASA), from water and methanol-water solutions, by help of a TOF-Q spectrometer equipped with electrospray ionization source, in the negative-ion mode. The structure and stability of the (H3N·SO3)n and [(H3N·SO3)n-H](-) (n = 1-6) were studied using DFT/B3LYP/aug-cc-pVDZ method. The ESI MS study evidenced that both singly and doubly charged clusters are formed when the acids are electrosprayed from water solutions; they may be described as [(H3N·SO3)n-zH](z-) where z = 1 or 2. The largest identified clusters are built of 20 monomers. The theoretical studies showed that formation of higher order (ASA)n aggregates in the gas phase is energetically profitable. In contrast with the gas phase, aqueous solution does not favor the formation of (ASA)n aggregates. The study led to the conclusion that the ASA clusters are formed in the gas phase under the experimental conditions of the mass spectrometer. A hypothetical mechanism concerning the formation of the doubly negatively charged anionic aggregates is discussed. The obtained data suggest that small (NH3·SO3)n aggregates may also contribute to formation of aerosols in heavily polluted atmospheres with relatively large NH3 concentration.

Auxin conjugated to fluorescent dyes--a tool for the analysis of auxin transport pathways.

Auxin is a small molecule involved in most processes related to plant growth and development. Its effect usually depends on the distribution in tissues and the formation of concentration gradients. Until now there has been no tool for the direct tracking of auxin transport at the cellular and tissue level; therefore the majority of studies have been based on various indirect methods. However, due to their various restrictions, relatively little is known about the relationship between various pathways of auxin transport and specific developmental processes. We present a new research tool: fluorescently labelled auxin in the form of a conjugate with two different fluorescent tracers, FITC and RITC, which allows direct observation of auxin transport in plant tissues. Chemical analysis and biological tests have shown that our conjugates have auxin-like biological activity and transport; therefore they can be used in all experimental systems as an alternative to IAA. In addition, the conjugates are a universal tool that can be applied in studies of all plant groups and species. The conjugation procedure presented in this paper can be adapted to other fluorescent dyes, which are constantly being improved. In our opinion, the conjugates greatly expand the possibilities of research concerning the role of auxin and its transport in different developmental processes in plants.

An attractive way of egg white protein by-product use for producing of novel anti-hypertensive peptides.

The aim of this study was to (i) examine how enzymatic hydrolysis with a non-commercially available proteinase of fig-leaf gourd fruit (Cucurbita ficifolia) increased the use value of egg white protein preparations, generated as byproducts in the industrial process of lysozyme and cystatin isolation from egg white, and (ii) evaluate the inhibition of angiotensin I-converting enzyme (ACE) by the obtained hydrolysates. Purification procedures including membrane filtration, gel filtration chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) led to the production of several peptide fractions. Two novel ovalbumin-derived tetrapeptides: SWVE (f 148-151) and DILN (f 86-89) with ACE inhibitory activity were obtained. Study of their inhibitory kinetics revealed a non-competitive binding mode, with an IC50 value against ACE of 33.88 and 73.44 µg for SWVE and DILN, respectively. Synthetic peptides which were designed on the basis of peptide SWVE were examined. A tripeptide sequence of SWV revealed the strongest ACE-inhibitory activity.

Differential zinc and DNA binding by partial peptides of human protamine HP2.

The Zn(II) binding by partial peptides of human protamine HP2: HP2(1-15); HP2(1-25), HP2(26-40), HP2(37-47), and HP2(43-57) was studied by circular dichroism (CD). Precipitation of a 20-mer DNA by these partial peptides and the effects of Zn(II) thereon were investigated using polyacrylamide gel electrophoresis (GE). The results of this study suggest that reduced HP2 (thiol groups intact) can bind Zn(II) at various parts of the molecule. In the absence of DNA, the primary Zn(II) binding site in reduced HP2 is located in the 37-47 sequence (involving Cys-37, His-39, His-43, and Cys-47), while in the presence of DNA, the strongest Zn(II) binding is provided by sequences 12-22 (by His-12, Cys-13, His-19, and His-22) and 43-57 (His-43, Cys-47, Cys-53, and His-57). In its oxidized form, HP2 can bind zinc through His residues of the 7-22 sequence. Zn(II) markedly enhances DNA binding by all partial peptides. These findings suggest that Zn(II) ions may be a regulatory factor for sperm chromatin condensation processes.

A two-process model describes the hydrogen exchange behavior of cytochrome c in the molten globule state with various extents of acetylation.

Acetylation of Lys residues of horse cytochrome c steadily stabilizes the molten globule state in 18 mM HCl as more Lys residues are acetylated [Goto and Nishikiori (1991) J. Mol. Biol. 222, 679-686]. The dynamic features of the molten globule state were characterized by hydrogen/deuterium exchange of amide protons, monitored by mass spectrometry as each deuteration increased the protein mass by 1 Da. Electrospray mass spectrometry enabled us to monitor simultaneously the exchange kinetics of more than seven species with a different number of acetyl groups. One to four Lys residue-acetylated cytochrome c showed almost no protection of the amide protons from rapid exchange. The transition from the unprotected to the protected state occurred between five and eight Lys residue-acetylated species. For species with more than nine acetylated Lys residues, the exchange kinetics were independent of the extent of acetylation, and 26 amide protons were protected at 60 min of exchange, indicating the formation of a rigid hydrophobic core with hydrogen-bonded secondary structures. The apparent transition to the protected state required a higher degree of acetylation than the conformational transition measured by circular dichroism, which had a midpoint at about four acetylated residues. This difference in the transitions suggested a two-process model in which the exchange occurs either from the protected folded state or from the unprotected unfolded state through global unfolding. On the basis of a two-process model and with the reported values of the exchange and stability parameters, we simulated the exchange kinetics of a series of acetylated cytochrome c species. The simulated kinetics reproduced the observed kinetics well, indicating validity of this model for hydrogen exchange of the molten globule state.

Immunosuppressory activity of the cyclodimeric peptide with RGD-sequences.

Our previous studies showed that the nonapeptide fragment of HLA-DQ of the sequence H-Thr-Pro-Gln-Arg-Gly-Asp-Val-Tyr-Thr-OH, located in the beta164-172 loop, strongly suppresses the humoral and cellular immune responses, while its shorter analogs, H-Arg-Gly-Asp-Val-OH, H-Arg-Gly-Asp-Val-Tyr-OH and H-Gln-Arg-Gly-Asp-Val-Tyr-OH show only a weak stimulatory activity in respect to the humoral immunological response. These fragments contain the Arg-Gly-Asp (RGD) sequence, known for its importance for cellular association phenomena. Based on the crystal structure of HLA-DR1, we also designed and synthesized a cyclic analog H-Cys-Arg-Gly-Asp-Val-Tyr-Cys-OH with restricted conformation, which strongly suppresses the immune response and selectively inhibits the alphavbeta3 integrin, suggesting that the mechanism of the immunosuppressory action of the peptide is associated with inhibition of the integrin. In this paper we present the design and synthesis of the cyclodimeric peptide, Arg-Gly-Asp-Arg-Gly-Asp, which is also known as a selective alphavbeta3 inhibitor. The synthesized peptide strongly suppresses both the humoral and cellular immune response. The results support our hypothesis that the immunomodulatory activity of HLA-DQ fragments may be connected with their interactions with some particular integrins on the cell surface.

Consecutive steps of phosphorylation affect conformation and DNA binding of the chironomus high mobility group A protein.

The high mobility group (HMG) proteins of the AT-hook family (HMGA) lie downstream in regulatory networks with protein kinase C, Cdc2 kinase, MAP kinase, and casein kinase 2 (CK2) as final effectors. In the cells of the midge Chironomus, almost all of the HMGA protein (cHMGA) is phosphorylated by CK2 at two adjacent sites. 40% of the protein population is additionally modified by MAP kinase. Using spectroscopic and protein footprinting techniques, we analyzed how individual and consecutive steps of phosphorylation change the conformation of an HMGA protein and affect its contacts with poly(dA-dT).poly(dA-dT) and a fragment of the interferon-beta promoter. We demonstrate that phosphorylation of cHMGA by CK2 alters its conformation and modulates its DNA binding properties such that a subsequent phosphorylation by Cdc2 kinase changes the organization of the protein-DNA complex. In contrast, consecutive phosphorylation by MAP kinase, which results in a dramatic change in cHMGA conformation, has no direct effect on the complex. Because the phosphorylation of the HMGA proteins attenuates binding affinity and reduces the extent of contacts between the DNA and protein, it is likely that this process mirrors the dynamics and diversity of regulatory processes in chromatin.

Conformational stability of six truncated cHMG1a proteins studied in their mixture by H/D exchange and electrospray ionization mass spectrometry.

The high mobility group (HMG) proteins are abundant non-histone components of eukaryotic chromatin. The presence of C-terminal acidic tails is a common feature of the majority of HMG proteins. Although the biological significance of the acidic domains is not clear, they are conferring conformational and metabolic stability to the proteins in vitro. Moreover, the length and net charge of the acidic tails affect the strength of HMG protein interaction with DNA. Synthesis of an insect HMG protein by standard recombinant technology in bacteria leads to a mixture of the intact protein (cHMG1a-(1-113) (I)) and a series of its degradation products truncated at the C tail: cHMG1a-(1-111) (II); cHMG1a-(1-110) (III); cHuMGla-(1-109) (IV); cHMG1a-(1-108) (V); cHMG1a-(1-107) (VI); cHMG1a-(1-106) (VII). The proteins differ from each other only by the number of amino-acid residues at the C-terminal tail. We used H/D exchange mass spectrometry to characterize the stability of the proteins directly in their mixture. The results show that the proteins I-V and VII have very similar conformations. The protein VI is less compact and exchanges its protons faster than the others. It may be concluded that the C-terminal tail influences the conformation of the cHMG1a protein and that individual residues in this part of the protein play a key role in its compactness.

Evaluation of high temperature glycation of proteins and peptides by electrospray ionization mass spectrometry.

Recently Boratynski & Roy (Glycoconjugate J., 1998, 15, 131) described a fast and convenient procedure for the synthesis of glycoconjugates. In the present study we used ESI-MS and circular dichroism as tools to analyze non-enzymatic glycation products of proteins and peptides. We discuss influence of reaction conditions on the rate of glycation of lysozyme. We analyze for the first time collision induced dissociation spectra of the obtained peptide conjugates.

Design, synthesis and biological evaluation of a new bridged immunosuppressor.

A bridged peptide with the sequence: H-Thr-Pro-Gln-Arg-Gly-Asp-Val-gamma-Abu-Asn-Asp-Gln-Glu-Glu-Thr-Thr-Gly-Val-Val-Ser-Thr-Pro-Leu-Ile-Arg-Asn-Gly-OH was designed to mimic the discontinuous epitope of the HLA-DQ molecule that might interact with CD4. The bridged peptide revealed distinct suppressory effect in the humoral immune response. This result supports our suggestion that the 164-172 region of the HLA-DQ molecule may enhance its interactions with coreceptors, possibly with CD4.

Inhibition of serine proteinases from human blood clotting system by squash inhibitor mutants.

A series of six CMTI I variants mutated in the P(2)-P(4)' region of the canonical binding loop were used to probe the role of single amino acid substitutions on binding to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X(a) and XII(a). The mutants were expressed as fusion proteins with the LE1413 hydrophobic polypeptide in Escherichia coli, purified from inclusion bodies, followed by cyanobromide cleavage and refolding. The mutants inhibited the proteinases with the association constants in the range 10(3)-10(9) M(-1). Inhibition of plasma kallikrein and factors X(a) and XII(a) could be improved up to 30-fold by single mutations. In contrast, neither of the introduced mutations increased inhibitory properties of CMTI I against plasmin. Additionally, using two inhibitors of natural origin, CMTI I (P(1) Arg) and CPTI II (P(1) Lys), we determined the effect of Lys-->Arg on binding to four proteinases. With the exception of plasmin (no effect), P(1) Arg resulted in up to 30-fold stronger binding than P(1) Lys.

New conformationally restricted analog of the immunosuppressory mini-domain of HLA-DQ and its biological properties.

Our previous studies revealed that the nonapeptide fragment of HLA-DQ located in the beta 164-172 loop of the Thr-Pro-Gln-Arg-Gly-Asp-Val-Tyr-Thr sequence suppresses the immune humoral and cellular responses [30]. Based on the crystal structure of HLA-class II molecules we designed and synthesized a cyclic analog with restricted conformation, cyclo(Suc-Thr-Pro-Gln-Arg-Gly-Asp-Val-Lys)-Thr-OH (Suc = succinyl) by reacting a Lys side chain with a succinylated N-terminus. The cyclization product more potently suppresses the cellular immune response than its linear counterparts and is efficiently cleaved by trypsin. The results indicate that the beta 164-172 loop may serve as a functional epitope on the HLA class II surface for intermolecular binding.

Immunosuppressory mini-regions of HLA-DP and HLA-DR.

Our previous studies showed, that the,TPQRGDVYT, QRGDVYT and RGDVYT fragments, located in the beta164-172 loop of HLA-DQ, strongly suppress the humoral and cellular immune response, while their shorter analogs, RGDV, RGDVY, and QRGDVY, show only weak stimulatory activity in respect to humoral immunological response. The fragments contain the RGDVY sequence that is analogous to thymopentin (pentapeptide RKDVY, an immune system activator) as well as the RGD sequence, known for its importance for cellular association phenomena. Based on the crystal structure of HLA-DR1, we also designed and synthesized a cyclic analog C*RGDVYC* (where C* indicates Cys participating in disulfide bridge) with restricted conformation, which strongly suppresses both humoral and cellular immune response. In the present study we synthesized and tested the immunological properties of the linear and cyclic HLA-DP and HLA-DR counterparts of all the above HLA-DQ fragments. Although the results show that the linear HLA-DP fragments possess moderate immunosuppressory potency, their conformationally restricted analog, C*QGDVYC*C shows a considerable suppression of both humoral and cellular immune response. The nonapeptide fragment of HLA-DR, VPRSGEVYT and particularly its cyclic analog C*SGEVYC*, are strong suppressors of the humoral response.

Constitutive phosphorylation of the acidic tails of the high mobility group 1 proteins by casein kinase II alters their conformation, stability, and DNA binding specificity.

The high mobility group (HMG) 1 and 2 proteins are the most abundant non-histone components of chromosomes. Here, we report that essentially the entire pool of HMG1 proteins in Drosophila embryos and Chironomus cultured cells is phosphorylated at multiple serine residues located within acidic tails of these proteins. The phosphorylation sites match the consensus phosphorylation site of casein kinase II. Electrospray ionization mass spectroscopic analyses revealed that Drosophila HMGD and Chironomus HMG1a and HMG1b are double-phosphorylated and that Drosophila HMGZ is triple-phosphorylated. The importance of this post-translational modification was studied by comparing some properties of the native and in vitro dephosphorylated proteins. It was found that dephosphorylation affects the conformation of the proteins and decreases their conformational and metabolic stability. Moreover, it weakens binding of the proteins to four-way junction DNA by 2 orders of magnitude, whereas the strength of binding to linear DNA remains unchanged. Based on these observations, we propose that the detected phosphorylation is important for the proper function and turnover rates of these proteins. As the occurrence of acidic tails containing canonical casein kinase II phosphorylation sites is common to diverse HMG and other chromosomal proteins, our results are probably of general significance.

The determination of thymopoietin conformation based on X-ray structure of a discontinuous thymopoietin-like motif of G-actin.

The biologically active conformation of thymopoietin, based on X-ray data reported for discontinuous thymopoietin-like motif of G-actin, is proposed. The conformation is compared with that resulting from the prediction made by the method of Chou & Fasman (Annu. Rev. Biochem. 47, 251-276, 1978) and Rost & Sander (Methods Enzymol. 266, 525-539, 1996).

Further investigations on thymopentin-like fragments of HLA-DQ and their analogs.

Recently we showed that the fragments of HLA-DQ with the Thr-Pro-Gln-Arg-Gly-Asp-Val-Tyr-Thr and Gln-Arg-Gly-Asp-Val-Tyr-Thr sequences strongly suppress the immune response, while their shorter analogs, Arg-Gly-Asp-Val, Arg-Gly-Asp-Val-Tyr and Gln-Arg-Gly-Asp-Val-Tyr, show very weak stimulatory activity with respect to humoral immunological response. The fragments contain the sequence which is very similar to thymopentin (pentapeptide Arg-Lys-Asp-Val-Tyr, an active fragment (32-36) of thymopoietin, an immune system activator produced in thymi), and at the same time contains the Arg-Gly-Asp (RGD) sequence, known as an inhibitor of adhesion processes. In the present study we found that a hexapeptide: Arg-Gly-Asp-Val-Tyr-Thr is the smallest size fragment of HLA-DQ having both cellular and humoral immunosuppressive activity. We also found that linear and cyclic fragments of HLA-DQ do not affect cell line production of various cytokines, what suggests that the mechanism of interactions of these peptides with the immunological system is different as compared with most other known immunosuppressors.

Immunological properties of the thymopentin-like fragments of HLA-DQ.

Class II human leukocyte antigens (HLA-II) are cell surface alpha beta heterodimers (M(r) approximately 60,000) that play a pivotal role in the immune response by presenting peptides derived from environmental antigens to the T-cell receptor. A 167-171 fragment of the beta 2-chain of the HLA-DQ molecule consists of the sequence RGDVY, which is very similar to thymopentin (pentapeptide RKDVY, an active fragment (32-36) of thymopoietin, an immune system activator produced in thymi), and at the same time contains the RGD sequence, known as an inhibitor of adhesion processes. We synthesized and investigated the immunomodulatory activity of series of peptide fragments of HLA-DQ containing thymopentin-like sequences. The results indicate that all synthesized peptides suppress the cellular immune response. However, RGDV, RGDVY and QRGDVY show very weak stimulatory activity in humoral immunological response tests. In contrast to the shorter peptides, the nonapeptide fragment of HLA-DQ, TPQRGDVYT, shows significant immunosuppressive activity in all tests. A possible role of these fragments of the polypeptide chain of HLA-DQ in the regulation of HLA functions is discussed.

The immunosuppressory and adhesive miniregion of the human major histocompatibility protein, human leukocyte antigen DQ.

Our previous studies showed that the nonapeptide fragment of human leukocyte antigen DQ with the TPQRGDVYT sequence strongly suppresses the immune response [Z. Szewczuk, I. Z. Siemion, and Z. Wieczorek (1996) Molecular Immunology, 33, 903-9081]. The fragment contains the RGDVY sequence, which is very similar to thymopentin (pentapeptide RKDVY, an active fragment (32-36) of thymopoietin, an immune system activator produced in thymi), and at the same time contains the RGD sequence, known as an inhibitor of adhesion processes. In the present study we tested an influence of the nonapeptide and its shorter fragments on binding the activated platelets and K562 cells to fibrinogen and fibronectin, respectively. We also designed and synthesized a cyclic thymopentin-like peptide. C*RGDVYC* (where C* indicates Cys participating in disulfide bridge) to restrict its conformation. The cyclization product strongly suppresses the humoral and cellular immune response and selectively inhibits the adhesion of K562 cells to fibronectin. The results are discussed in the light of CD conformational studies.