Conformational study of N-methylated alanine peptides and design of Aẞ inhibitor.

N-Methylation increases the proteolytic stability of peptides and leads to improved pharmacological and increased nematicidal property against plant pathogens. In this study, the quantum mechanical and molecular dynamic simulation approaches were used to investigate conformational behavior of peptides containing only N-methylated alanine (NMeAla) residues and N-methylated alanine and alanine residues at alternate positions. The amide bond geometry was found to be trans and the poly NMeAla peptides were shown to populate in the helical structure without hydrogen bond with , values of ~ 0, 90˚ stabilized by carbonyl-carbonyl interactions. Molecular dynamic simulations in water/methanol revealed the formation of β-strand structure, irrespective of the starting geometry due to the interaction of solvent molecules with the carbonyl groups of peptide backbone. Analysis of simulation results as a function of time suggested that the opening of helical structure without hydrogen bond started from C-terminal. Conformational behavior of peptides containing N-MeAla and Ala was used to design Ab peptide inhibitor and the model tetrapeptide Ac-Ala-NMeAla-Ala-NHMe in the β-strand structure was shown to interact with the hydrophobic stretch of Aβ15-42 peptide.

Comparison of two peptide radiotracers for prostate carcinoma targeting

OBJECTIVES: Scintigraphy is generally not the first choice treatment for prostate cancer, although successful studies using bombesin analog radiopeptides have been performed. Recently, a novel peptide obtained using a phage display library demonstrated an affinity for prostate tumor cells. The aim of this study was to compare the use of a bombesin analog to that of a phage display library peptide (DUP-1) radiolabeled with technetium-99m for the treatment of prostate carcinoma. The peptides were first conjugated to S-acetyl-MAG3 with a 6-carbon spacer, namely aminohexanoic acid. METHODS: The technetium-99m labeling required a sodium tartrate buffer. Radiochemical evaluation was performed using ITLC and was confirmed by high-performance liquid chromatography. The coefficient partition was determined, and in vitro studies were performed using human prostate tumor cells. Biodistribution was evaluated in healthy animals at various time points and also in mice bearing tumors. RESULTS: The radiochemical purity of both radiotracers was greater than 95 percent. The DUP-1 tracer was more hydrophilic (log P = -2.41) than the bombesin tracer (log P = -0.39). The biodistribution evaluation confirmed this hydrophilicity by revealing the greater kidney uptake of DUP-1. The bombesin concentration in the pancreas was greater than that of DUP-1 due to specific gastrin-releasing peptide receptors. Bombesin internalization occurred for 78.32 percent of the total binding in tumor cells. The DUP-1 tracer showed very low binding to tumor cells during the in vitro evaluation, although tumor uptake for both tracers was similar. The tumors were primarily blocked by DUP1 and the bombesin radiotracer primarily targeted the pancreas. CONCLUSION: Further studies with the radiolabeled DUP-1 peptide are recommended. With further structural changes, this molecule could become an efficient alternative tracer for prostate tumor diagnosis.

Effect of RGD-modified silk material on the adhesion and proliferation of bone marrow-derived mesenchymal stem cells / 华中科技大学学报（医学）（英德文版）

In order to investigate the effect of Arg-Gly-Asp (RGD) peptide-modified silk biomaterial on the adhesion and proliferation of bone marrow-derived mesenchymal stem cells (MSCs), MSCs of third generation were seeded onto the surface of RGD-decorated silk (silk-RGD group), silk alone (silk group) or tissue culture plate (TCP group). After incubation for 4 or 12 h, MSCs were examined quantitatively by using precipitation method for cell attachment. The cell proliferation, which was defined as cell density, was compared among the three groups after culture for 1, 2, 3, and 4 days. Cell skeleton, which was labeled fluorescently, was observed under laser confocal microscope after 24 h of culture. The results showed that cell adhesion rate in silk-RGD group was higher than in silk group (P0.05). There were no significant differences in the cell proliferation among the three groups at different time points (P>0.05 for all). Laser confocal microscopy revealed that in silk-RGD group, MSCs, strongly fluorescently stained, spread fully, with stress fibers clearly seen, while in silk group, actin filaments were sparsely aligned and less stress fibers were found. It was concluded that RGD peptide could improve the adhesion of MSCs to the silk scaffold, but had no impact on the proliferation of the cells.

Modeling studies on phospholipase A2-inhibitor complexes.

Phospholipase A2 (PLA2) is a ubiquitous enzyme that specifically catalyzes hydrolysis of membrane phospholipids to produce lysophospholipids and free fatty acid, namely arachidonic acid, which provides substrate for eicosanoids biosynthesis. Thus, the compounds inhibiting PLA2 have been implicated as potential therapeutic agents in treatment of inflammation related diseases. Plant and marine organisms serve as sources of compounds that act as potential therapeutic agents for treatment of various diseases. The present study reveals the relationship between the structure and function of the medicinally important herbal compounds (acalyphin, chlorogenic acid, stigmasterol, curcumin and tectoridin) and marine compounds (gracilin A and aplysulphurin A). To understand the binding mechanisms of these compounds, molecular modeling studies has been performed with Russell's viper and bovine pancreatic PLA2 as target molecules using molecular operating environment (MOE) software. These compounds show favorable interactions with the amino acid residues at the active site of Russell's viper and bovine pancreatic PLA2, thereby substantiating their proven efficacy as anti-inflammatory compounds and antidotes.

Molecular vibrations and normal modes in L-prolyl-glycyl-glycine using Wilson GF matrix method.

Collagen is one of the most important proteins containing mostly proline hydroxyproline and glycine. In collagen, approximately 33 percent of the amino acid residues are glycine and they occur at every third position, whereas remaining percentage is constituted by mainly proline or hydroxyproline and some part by alanine etc. having no definite positional placement in the chain. Thus, a study of conformation of proline and glycine containing dipeptides and tripeptides is important for understanding the conformation of collagen as a sequence of its constituent amino acids. In the present communication, we have studied spectral features of L-proline, L-prolyl-glycine (PG), L-prolyl-alanine (PA), L-glycylglycine (GG), Collagen and L-prolyl-glycyl-glycine (PGG). We have carried out detailed normal mode analysis of only PGG, because interpretation of spectra of other proline and glycine containing peptides can be treated as derivatives of this molecule. Urey-Bradley force field, which involves non-bonded interactions in the gem and cis configurations is used for calculation of normal modes. The "best-fit" set of constants are generated for PGG.

Protein local conformations arise from a mixture of Gaussian distributions.

The classical approaches for protein structure prediction rely either on homology of the protein sequence with a template structure or on ab initio calculations for energy minimization. These methods suffer from disadvantages such as the lack of availability of homologous template structures or intractably large conformational search space, respectively. The recently proposed fragment library based approaches first predict the local structures,which can be used in conjunction with the classical approaches of protein structure prediction. The accuracy of the predictions is dependent on the quality of the fragment library. In this work, we have constructed a library of local conformation classes purely based on geometric similarity. The local conformations are represented using Geometric Invariants, properties that remain unchanged under transformations such as translation and rotation, followed by dimension reduction via principal component analysis. The local conformations are then modeled as a mixture of Gaussian probability distribution functions (PDF). Each one of the Gaussian PDF's corresponds to a conformational class with the centroid representing the average structure of that class. We find 46 classes when we use an octapeptide as a unit of local conformation. The protein 3-D structure can now be described as a sequence of local conformational classes. Further, it was of interest to see whether the local conformations can be predicted from the amino acid sequences. To that end,we have analyzed the correlation between sequence features and the conformational classes.

Using bradykinin-potentiating peptide structures to develop new antihypertensive drugs

Angiotensin I-converting enzyme (ACE) is a dipeptidyl-carboxypeptidase expressed in endothelial, epithelial and neuroepithelial cells. It is composed of two domains, known as N- and C-domains, and it is primarily involved in blood pressure regulation. Although the physiological functions of ACE are not limited to its cardiovascular role, it has been an attractive target for drug design due to its critical role in cardiovascular and renal disease. We examined natural structures based on bradykinin-potentiating peptides (BPPs) extracted from Bothrops jararaca venom for ACE inhibition. Modeling, docking and molecular dynamics were used to study the conserved residues in the S2’, S1’ and S1 positions that allow enzyme-substrate/inhibitor contacts. These positions are conserved in other oligopeptidases, and they form tight and non-specific contacts with lisinopril, enalapril and BPP9a inhibitors. The only specific inhibitor for human somatic ACE (sACE) was BPP9a, which is instable in the N-sACE-BPP9a complex due to repulsive electrostatic interactions between Arg P4-Arg 412 residues. Specificity for the C-terminal domain in human sACE inhibition was confirmed by electrostatic interaction with the Asp 1008 residue. Peptide-like BPP structures, naturally developed by snakes across millions of years of evolution, appear to be good candidates for the development of domain-selec tive ACE inhibitors with high stability and improved pharmacological profiles.

Conformational features of a hexapeptide model Ac-TGAAKA-NH2 corresponding to a hydrated alpha helical segment from glyceraldehyde 3-phosphate dehydrogenase: implications for the role of turns in helix folding.

Recent analysis of alpha helices in protein crystal structures, available in literature, revealed hydrated alpha helical segments in which, water molecule breaks open helix 5-->1 hydrogen bond by inserting itself, hydrogen bonds to both C=O and NH groups of helix hydrogen bond without disrupting the helix hydrogen bond, and hydrogen bonds to either C=O or NH of helix hydrogen bond. These hydrated segments display a variety of turn conformations and are thought to be 'folding intermediates' trapped during folding-unfolding of alpha helices. A role for reverse turns is implicated in the folding of alpha helices. We considered a hexapeptide model Ac-1TGAAKA6-NH2 from glyceraldehyde 3-phosphate dehydrogenase, corresponding to a hydrated helical segment to assess its role in helix folding. The sequence is a site for two 'folding intermediates'. The conformational features of the model peptide have been investigated by 1H 2D NMR techniques and quantum mechanical perturbative configuration interaction over localized orbitals (PCILO) method. Theoretical modeling largely correlates with experimental observations. Based upon the amide proton temperature coefficients, the observed d alpha n(i, i + 1), d alpha n(i, i + 2), dnn(i, i + 1), d beta n(i, i + 1) NOEs and the results from theoretical modeling, we conclude that the residues of the peptide sample alpha helical and neck regions of the Ramachandran phi, psi map with reduced conformational entropy and there is a potential for turn conformations at N and C terminal ends of the peptide. The role of reduced conformational entropy and turn potential in helix formation have been discussed. We conclude that the peptide sequence can serve as a 'folding intermediate' in the helix folding of glyceraldehyde 3-phosphate dehydrogenase.

Effect of synthetic nonapeptide (Thr-Cys-Ser-Val-Ser-Glu-Trp-Gly-Ile) on ovarian follicular development in the bull frog Rana tigrina (Daud).

Effect of synthetic nonapeptide (Thr-Cys-Ser-Val-Ser-Glu-Trp-Gly-Ile) representing the amino acid sequence 86-94 of human seminal plasma was studied on the ovarian follicular growth in the bullfrog R. tigrina during preparatory phase of reproductive cycle. Daily (except on Sundays) injections of 10 micrograms nonapeptide for one month caused a significant increase in ovarian weight and number of second growth phase (SGP) or vitellogenic oocytes. The results suggest that the nonapeptide is biologically active in amphibians also.

Purification and characterization of an alkaline proteinase from kidney cortex lysosomes.

An alkaline proteinase was purified to apparent homogeneity from buffalo (Bubalus bubalis) kidney cortex lysosomes by affinity chromatography on STI sepharose 4B and gel filtration over Sephadex G-100. The molecular weight of the enzyme was 17,000 and 21,000 by gel filtration and SDS/PAGE respectively. The purified enzyme was optimally active at pH 8.5-9.0 at 50 degrees C and hydrolysed synthetic substrates of chymotrypsin but not those of elastase or trypsin. It was inhibited by serine proteinase inhibitors like soybean trypsin inhibitor, limabean trypsin inhibitor and phenylmethyl sulphonyl fluoride. Immunologically, the enzyme was similar to chymotrypsin. The amino acid composition showed high content of acidic amino acids. This protein was detected in kidney, liver, spleen, pancreas and heart.

Solution conformation of a model hexapeptide containing RGD sequence.

The solution conformation of a model hexapeptide Asp-Arg-Gly-Asp-Ser-Gly (DRGDSG) containing the RGD sequence has been studied in DMSO-d6 as well as in aqueous solution (H2O:D2O/90:10%) by 1H NMR spectroscopy. The unambiguous identification of spin systems of various amino acid residues and sequence specific assignment of all proton resonances was achieved by a combination of two dimensional COSY and NOESY experiments. The temperature coefficient data of the amide proton chemical shifts in conjunction with the vicinal coupling constants, i.e. 3JNH-C alpha H, NOESY and ROESY results indicate that the peptide in both the solvents exists in a blend of conformers with beta-sheet like extended backbone structure and folded conformations. The folded conformers do not appear to be stabilised by intramolecular hydrogen bonding. Our results are consistent with the flexibility of RGD segment observed in the NMR studies on the protein echistatin containing the RGD motif (references 23-25).

A 500 MHz proton NMR study of interaction of tripeptides Lys-Tyr-Lys and Lys-Phe-Lys with deoxydinucleotide d-CpG.

The binding of di- and tetranucleotides with tri- and tetrapeptides containing Tyr, Trp, Phe having lysine on both ends has been studied using a 500 MHz proton NMR. The results show that d-CpG exists as a right-handed B-DNA structure with both sugars in 01'-endo sugar conformation and glycosidic bond angle as in anti domain. On binding to tripeptide Lys-Tyr-Lys, the Tyr ring protons shift upfield by 0.015 ppm at 285 degrees K, while the conformation of d-CpG remains unchanged. Change in chemical shift of Tyr and nucleotide protons decreases with temperature. This upfield shift is attributed to stacking with bases/base-pairs. The presence of intermolecular NOE's also supports this. Results of binding of d-CpG to Lys-Phe-Lys are similar to those with Lys-Tyr-Lys except that the chemical shift changes occur to a lesser extent. On comparing the results obtained with three different peptides, it is found that interaction decreases in the order Trp > Tyr > Phe which is similar to that found by theoretical energy calculations (reported elsewhere) and fluorescence measurements. The results also exhibit a specificity in recognition of these amino acid residues by dinucleotides.

Peptide transport across the animal cell plasma membrane: recent developments.

Transfer of intact peptides across the plasma membrane of animal cells, especially in the small intestine and the kidney, is a well established phenomenon. This process plays an important role in the maintenance of protein nutrition. Evidence is accumulating which suggests that the process may also have a great potential for pharmacological and clinical applications. It is therefore important to understand various aspects of peptide transport such as its function, chemical nature of the transport protein and its gene, the operational mechanisms and their regulation, and the relevance of the transport system to health and disease. Recent years have witnessed considerable progress in the field. The driving force for the transport system has been identified as the proton motive force which makes the system unique and distinct from the majority of solute transport systems in animal cells which are driven by a sodium motive force. A great deal is now known on the chemical nature of the active site. The protein responsible for the transport process in the small intestine has been purified and characterized. The system has been successfully expressed in its functional form in Xenopus laevis oocytes by microinjection into the oocytes of poly(A)+ mRNA isolated from intestinal mucosal cells. There is no doubt that the coming years will bring even more exciting information on the transport system, especially in areas such as hormonal regulation, clinical applicability and cloning, and characterization of the gene encoding the transport system.