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1. |
Understanding interactions ofgastric inhibitory polypeptide(GIP) with its G‐protein coupled receptor through NMR and molecular modeling |
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Journal of Peptide Science,
Volume 13,
Issue 5,
2007,
Page 287-300
Alpeshkumar K. Malde,
Sudha S. Srivastava,
Evans C. Coutinho,
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摘要:
AbstractGastric inhibitory polypeptide (GIP, or glucose‐dependent insulinotropic polypeptide) is a 42‐amino acid incretin hormone moderating glucose‐induced insulin secretion. Antidiabetic therapy based on GIP holds great promise because of the fact that its insulinotropic action is highly dependent on the level of glucose, overcoming the sideeffects of hypoglycemia associated with the current therapy of Type 2 diabetes. The truncated peptide, GIP(1–30)NH2, has the same activity as the full length native peptide. We have studied the structure of GIP(1–30)NH2and built a model of its G‐protein coupled receptor (GPCR). The structure of GIP(1–30)NH2in DMSO‐d6and H2O has been studied using 2D NMR (total correlation spectroscopy (TOCSY), nuclear overhauser effect spectroscopy (NOESY), double quantum filtered‐COSY (DQF‐COSY),13C‐heteronuclear single quantum correlation (HSQC) experiments, and its conformation built by MD simulations with the NMR data as constraints. The peptide in DMSO‐d6exhibits an α‐helix between residues Ile12 and Lys30 with a discontinuity at residues Gln19 and Gln20. In H2O, the α‐helix starts at Ile7, breaks off at Gln19, and then continues right through to Lys30. GIP(1–30)NH2has all the structural features of peptides belonging to family B1 GPCRs, which are characterized by a coil at theN‐terminal and a longC‐terminal α‐helix with or without a break. A model of the seven transmembrane (TM) helices of the GIP receptor (GIPR) has been built on the principles of comparative protein modeling, using the crystal structure of bovine rhodopsin as a template. TheN‐terminal domain of GIPR has been constructed from the NMR structure of theN‐terminal of corticoptropin releasing factor receptor (CRFR), a family B1 GCPR. The intra and extra cellular loops and theC‐terminal have been modeled from fragments retrieved from the PDB. On the basis of the experimental data available for some members of family B1 GPCRs, four pairs of constraints between GIP(1–30)NH2and its receptor were used in the FTDOCK program, to build the complete model of the GIP(1–30)NH2: GIPR complex. The model can rationalize the various experimental observations including the potency of the truncated GIP peptide. This work is the first complete model at the atomic level of GIP(1–30)NH2and of the complex with its GPCR. Copyright © 200
ISSN:1075-2617
DOI:10.1002/psc.839
出版商:John Wiley&Sons, Ltd.
年代:2007
数据来源: WILEY
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2. |
Interaction of S413‐PV cell penetrating peptide with model membranes: relevance to peptide translocation across biological membranes |
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Journal of Peptide Science,
Volume 13,
Issue 5,
2007,
Page 301-313
Miguel Mano,
Ana Henriques,
Artur Paiva,
Manuel Prieto,
Francisco Gavilanes,
Sérgio Simões,
Maria C. Pedroso De Lima,
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摘要:
AbstractCell penetrating peptides (CPPs) have been successfully used to mediate the intracellular delivery of a wide variety of molecules of pharmacological interest bothin vitroandin vivo, although the mechanisms by which the cellular uptake occurs remain unclear and controversial. Following our previous work demonstrating that the cellular uptake of the S413‐PV CPP occurs mainly through an endocytosis‐independent mechanism, we performed a detailed biophysical characterization of the interaction of this peptide with model membranes. We demonstrate that the interactions of the S413‐PV peptide with membranes are essentially of electrostatic nature. As a consequence of its interaction with negatively charged model membranes, the S413‐PV peptide becomes buried into the lipid bilayer, which occurs concomitantly with significant peptide conformational changes that are consistent with the formation of a helical structure. Comparative studies using two related peptides demonstrate that the conformational changes and the extent of cell penetration are dependent on the peptide sequence, indicating that the helical structure acquired by the S413‐PV peptide is relevant for its nonendocytic uptake. Overall, our data suggest that the cellular uptake of the S413‐PV CPP is a consequence of its direct translocation through cell membranes, following conformational changes induced by peptide‐membrane interactions. Copyright © 2007 European Peptide Society and John W
ISSN:1075-2617
DOI:10.1002/psc.842
出版商:John Wiley&Sons, Ltd.
年代:2007
数据来源: WILEY
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3. |
The sequence TGAAKAVALVL from glyceraldehyde‐3‐phosphate dehydrogenase displays structural ambivalence and interconverts between α‐helical and β‐hairpin conformations mediated by collapsed conformational states |
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Journal of Peptide Science,
Volume 13,
Issue 5,
2007,
Page 314-326
Sunita Patel,
Petety V. Balaji,
Yellamraju U. Sasidhar,
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摘要:
AbstractThe peptide TGAAKAVALVL from glyceraldehyde‐3‐phosphate dehydrogenase adopts a helical conformation in the crystal structure and is a site for two hydrated helical segments, which are thought to be helical folding intermediates. Overlapping sequences of four to five residues from the peptide, sample both helical and strand conformations in known protein structures, which are dissimilar to glyceraldehyde‐3‐phosphate dehydrogenase suggesting that the peptide may have a structural ambivalence. Molecular dynamics simulations of the peptide sequence performed for a total simulation time of 1.2 µs, starting from the various initial conformations using GROMOS96 force field under NVT conditions, show that the peptide samples a large number of conformational forms with transitions from α‐helix to β‐hairpin and vice versa. The peptide, therefore, displays a structural ambivalence. The mechanism from α‐helix to β‐hairpin transition and vice versa reveals that the compact bends and turns conformational forms mediate such conformational transitions. These compact structures including helices and hairpins have similar hydrophobic radius of gyration (Rgh) values suggesting that similar hydrophobic interactions govern these conformational forms. The distribution of conformational energies is Gaussian with helix sampling lowest energy followed by the hairpins and coil. The lowest potential energy of the full helix may enable the peptide to take up helical conformation in the crystal structure of the glyceraldehyde‐3‐phosphate dehydrogenase, even though the peptide has a preference for hairpin too. The relevance of folding and unfolding events observed in our simulations to hydrophobic collapse model of protein folding are discussed. Copyright © 2007 European Peptide Society
ISSN:1075-2617
DOI:10.1002/psc.843
出版商:John Wiley&Sons, Ltd.
年代:2007
数据来源: WILEY
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4. |
Conformational solution studies of the anti‐microbial temporin A retro‐analogues by using NMR spectroscopy |
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Journal of Peptide Science,
Volume 13,
Issue 5,
2007,
Page 327-333
Wojciech Kamysz,
Beata Mickiewicz,
Katarzyna Greber,
Sylwia Rodziewicz‐MotowidŁo,
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摘要:
AbstractTemporin A (TA) is a small, basic and highly hydrophobic peptide, isolated from the skin of the European red frog,Rana temporaria. The TA (FLPLIGRVLSGIL‐NH2) displays a broad spectrum of anti‐microbial activity against Gram‐positive bacteria and fungiCandida albicans. In this study we investigate the solution structure of two TA retro‐analogues, (6‐1)(7–13)‐TA (GILPLFRVLSGIL‐NH2) and retro‐TA (LIGSLVRGILPLF‐NH2) by using nuclear magnetic resonance (NMR). The 3D solution structure of the analogues was established by using inter‐proton distances and vicinal coupling constants in theSimulated Annealing(SA) calculations (XPLOR program). The NMR conformational studies show the existence of the helical structure in the middle part of the (6‐1)(7–13)‐TA peptide and an unordered structure of the retro‐TA analogue under the D3‐TFE/H2O (3:7, v/v) conditions. Our investigations have shown that the hydrophobic cluster atN‐terminus with the Pro amino acid residue in position 3 or 4, the helical structure and the amphipathic character of the peptide are responsible for the anti‐microbial activity of the TA analogues. Copyright © 2007 European Pept
ISSN:1075-2617
DOI:10.1002/psc.844
出版商:John Wiley&Sons, Ltd.
年代:2007
数据来源: WILEY
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5. |
The use of Fmoc‐Lys(Pac)‐OH and penicillin G acylase in the preparation of novel semisynthetic insulin analogs |
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Journal of Peptide Science,
Volume 13,
Issue 5,
2007,
Page 334-341
Lenka Žáková,
Daniel Zyka,
Jan Ježek,
Ivona Hančlová,
Miloslav Šanda,
Andrzej M. Brzozowski,
Jiří Jiráček,
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摘要:
AbstractIn this paper, we present the detailed synthetic protocol and characterization of Fmoc‐Lys(Pac)‐OH, its use for the preparation of octapeptides H‐Gly‐Phe‐Tyr‐N‐MePhe‐Thr‐Lys(Pac)‐Pro‐Thr‐OH and H‐Gly‐Phe‐Phe‐His‐Thr‐Pro‐Lys(Pac)‐Thr‐OH by solid‐phase synthesis, trypsin‐catalyzed condensation of these octapeptides with desoctapeptide(B23‐B30)‐insulin, and penicillin G acylase catalyzed cleavage of phenylacetyl (Pac) group fromNε‐amino group of lysine to give novel insulin analogs [TyrB25,N‐MePheB26,LysB28,ProB29]‐insulin and [HisB26]‐insulin. These new analogs display 4 and 78% binding affinity respectively to insulin receptor in rat adipose membranes. Copyrigh
ISSN:1075-2617
DOI:10.1002/psc.847
出版商:John Wiley&Sons, Ltd.
年代:2007
数据来源: WILEY
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6. |
Peptides and proteins in a confined environment: NMR spectra at natural isotopic abundance |
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Journal of Peptide Science,
Volume 13,
Issue 5,
2007,
Page 342-347
Annalisa Pastore,
Severo Salvadori,
Piero Andrea Temussi,
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摘要:
AbstractConfinement of proteins and peptides in a small inert space mimics the natural environment of the cell, allowing structural studies in conditions that stabilize folded conformations. We have previously shown that confinement in polyacrylamide gels (PAGs) is sufficient to induce a change in the viscosity of the aqueous solution without changing the composition and temperature of the solvent. The main limitation of a PAG to run NMR experiments in a confined environment is the need for labelling the peptides. Here we report the use of the agarose gel to run the NMR spectra of proteins and peptides. We show that agarose gels are completely transparent in NMR experiments, relieving the need for labelling. Although it is necessary to expose biomolecules to fairly high temperatures during sample preparation, we believe that this is not generally an obstacle to the study of peptides, and found that the method is also compatible with temperature‐resistant proteins. The mesh of agarose gels is too wide for direct effects of confinement on the stability of proteins but confinement can be easily exploited to interact the proteins with other reagents, including crowding macromolecules that can eventually lead to fold stabilization. The use of these gels is ideally suited for low‐temperature studies; we show that a very flexible peptide at subzero temperatures is stabilized into a well‐folded conformation. Copyright © 2007 European Peptide Society and John Wiley&Son
ISSN:1075-2617
DOI:10.1002/psc.848
出版商:John Wiley&Sons, Ltd.
年代:2007
数据来源: WILEY
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7. |
Activity and stability of human kallikrein‐2‐specific linear and cyclic peptide inhibitors |
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Journal of Peptide Science,
Volume 13,
Issue 5,
2007,
Page 348-353
Miikka Pakkala,
Can Hekim,
Pasi Soininen,
Jari Leinonen,
Hannu Koistinen,
Janne Weisell,
Ulf‐HÅkan Stenman,
Jouko Vepsäläinen,
Ale Närvänen,
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摘要:
AbstractHuman glandular kallikrein (KLK2) is a highly prostate‐specific serine protease, which is mainly excreted into the seminal fluid, but part of which is also secreted into circulation from prostatic tumors. Since the expression level of KLK2 is elevated in aggressive tumors and it has been suggested to mediate the metastasis of prostate cancer, inhibition of the proteolytic activity of KLK2 is of potential therapeutic value. We have previously identified several KLK2‐specific linear peptides by phage display technology. Two of its synthetic analogs, A R R P A P A P G (KLK2a) and G A A R F K V W W A A G (KLK2b), show specific inhibition of KLK2 but their sensitivity to proteolysisin vivomay restrict their potential use as therapeutic agents. In order to improve the stability of the linear peptides forin vivouse, we have prepared cyclic analogs and compared their biological activity and their structural stability. A series of cyclic variants with cysteine bridges were synthesized. Cyclization inactivated one peptide (KLK2a) and its derivatives, while the other peptide (KLK2b) and its derivatives remained active. Furthermore, backbone cyclization of KLK2b improved significantly the resistance against proteolysis by trypsin and human plasma. Nuclear magnetic resonance studies showed that cyclization of the KLK2b peptides does not make the structures more rigid. In conclusion, we have shown that backbone cyclization of KLK2 inhibitory peptides can be used to increase stability without losing biological activity. This should render the peptides more useful forin vivoapplications, such as tumor imaging and prostate cancer targeting. Copyright © 2007 European Peptide Society and John Wiley&Sons,
ISSN:1075-2617
DOI:10.1002/psc.849
出版商:John Wiley&Sons, Ltd.
年代:2007
数据来源: WILEY
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8. |
Application of gel‐phase19F NMR spectroscopy for optimization of solid‐phase synthesis of a hydrophobic peptide from the signal sequence of the mucin MUC1 |
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Journal of Peptide Science,
Volume 13,
Issue 5,
2007,
Page 354-361
Maciej Pudelko,
Jan Kihlberg,
Mikael Elofsson,
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摘要:
AbstractThis paper describes the manual Fmoc/t‐Bu solid‐phase synthesis of a difficult nine‐residue hydrophobic peptide LLLLTVLTV from one of the signal sequences that flank the tandem repeat of the mucin MUC1. Gel‐phase19F NMR spectroscopy was used as a straightforward method for optimization of the solid‐phase synthesis. Different approaches were applied for comparative studies. The strategy based on modified solid‐phase conditions using DIC/HOAt for coupling, DBU for Fmoc deprotection, and the incorporation of the pseudo proline dipeptide Fmoc‐Leu‐Thr(ψMe,Mepro)‐OH as a backbone‐protecting group was found to be superior according to gel‐phase19F NMR spectroscopy. Implementation of the optimized Fmoc protocol enabled an effective synthesis of signal peptide LLLLTVLTV. Copyright © 2007 European Peptide Society a
ISSN:1075-2617
DOI:10.1002/psc.850
出版商:John Wiley&Sons, Ltd.
年代:2007
数据来源: WILEY
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