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Basic Residues (basic + residue)
Selected AbstractsPeptide T revisited: conformational mimicry of epitopes of anti-HIV proteinsJOURNAL OF PEPTIDE SCIENCE, Issue 4 2001Delia Picone Abstract Peptide T (ASTTTNYT), a fragment corresponding to residues 185,192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C -terminal pentapeptide, and SAR studies showing that the C -terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations, albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N -terminus and a basic residue at the C -terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19,26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C -terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd. [source] A double mutation of Escherichia coli 2C -methyl- d -erythritol-2,4-cyclodiphosphate synthase disrupts six hydrogen bonds with, yet fails to prevent binding of, an isoprenoid diphosphateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2005Tanja Sgraja The essential enzyme 2C -methyl- d -erythritol-2,4-cyclodiphosphate (MECP) synthase, found in most eubacteria and the apicomplexan parasites, participates in isoprenoid-precursor biosynthesis and is a validated target for the development of broad-spectrum antimicrobial drugs. The structure and mechanism of the enzyme have been elucidated and the recent exciting finding that the enzyme actually binds diphosphate-containing isoprenoids at the interface formed by the three subunits that constitute the active protein suggests the possibility of feedback regulation of MECP synthase. To investigate such a possibility, a form of the enzyme was sought that did not bind these ligands but which would retain the quaternary structure necessary to create the active site. Two amino acids, Arg142 and Glu144, in Escherichia coli MECP synthase were identified as contributing to ligand binding. Glu144 interacts directly with Arg142 and positions the basic residue to form two hydrogen bonds with the terminal phosphate group of the isoprenoid diphosphate ligand. This association occurs at the trimer interface and three of these arginines interact with the ligand phosphate group. A dual mutation was designed (Arg142 to methionine and Glu144 to leucine) to disrupt the electrostatic attractions between the enzyme and the phosphate group to investigate whether an enzyme without isoprenoid diphosphate could be obtained. A low-resolution crystal structure of the mutated MECP synthase Met142/Leu144 revealed that geranyl diphosphate was retained despite the removal of six hydrogen bonds normally formed with the enzyme. This indicates that these two hydrophilic residues on the surface of the enzyme are not major determinants of isoprenoid binding at the trimer interface but rather that hydrophobic interactions between the hydrocarbon tail and the core of the enzyme trimer dominate ligand binding. [source] Alanine screening of the intracellular loops of the human bradykinin B2 receptor , effects on receptor maintenance, G protein activation and internalizationFEBS JOURNAL, Issue 13 2009Alexander Faussner The bradykinin B2 receptor is coupled to G protein Gq/11 and becomes sequestered into intracellular compartments after activation. To more closely define the receptor sequences involved in these processes and their functions, we systematically mutated all three intracellular loops (ICLs), either as point mutations or in groups of three to five amino acids to Ala, obtaining a total of 14 mutants. All constructs were stably expressed in HEK 293 cells and, with the exception of triple mutant DRY , AAA, retained the ability to specifically bind [3H]bradykinin. The binding affinities at 4 or 37 °C of several mutants differed considerably from those determined for the wild-type receptor, indicating an allosteric connection between the conformation of the binding site and that of the ICLs. Mutations in ICL-1 strongly reduced surface expression without affecting G protein signaling or [3H]bradykinin internalization. Two cluster mutants in the middle of ICL-2 containing basic residues displayed considerably reduced potencies, whereas two mutations in ICL-3 resulted in receptor conformations that were considered to be semi-active, based on the observation that they responded with phosphoinositide hydrolysis to compounds normally considered to be antagonists. This, and the fact that a cluster mutant at the C-terminal end of ICL-3 was signaling incompetent, hint at the involvement of ICL-2 and ICL-3 in Gq/11 activation, albeit with different functions. None of the mutants displayed reduced ligand-induced receptor internalization, indicating that the loops are not essential for this process. No conclusion could be drawn, however, with regard to the role of the DRY sequence, as the corresponding triplet mutation lacked binding capability. [source] The porcine trophoblastic interferon-,, secreted by a polarized epithelium, has specific structural and biochemical propertiesFEBS JOURNAL, Issue 11 2002Avrelija Cenci At the time of implantation in the maternal uterus, the trophectoderm of the pig blastocyst is the source of a massive secretion of interferon-gamma (IFN-,), together with lesser amounts of IFN-,, a unique species of type I IFN. This trophoblastic IFN-, (TrIFN-,) is an unprecedented example of IFN-, being produced spontaneously by an epithelium. We therefore studied some of its structural and biochemical properties, by comparison with pig IFN-, from other sources, either natural LeIFN-, (from adult leucocytes), or recombinant. Biologically active TrIFN-, is a dimeric molecule, of which monomers are mainly composed of a truncated polypeptide chain with two glycotypes, unlike LeIFN-, which is formed of at least two polypeptide chains and four glycotypes. TrIFN-, collected in the uterus lumen was enzymatically deglycosylated and analysed by mass spectrometry (MALDI-TOF). The data revealed that the more abundant polypeptide has a mass of 14.74 kDa, corresponding to a C-terminal cleavage of 17 residues from the expected 143-residue long mature sequence. A minor polypeptide, with a mass of 12.63 kDa, corresponds to a C-terminal truncation of 36 amino acids. MALDI-TOF analysis of tryptic peptides from the glycosylated molecule(s) identifies a single branched carbohydrate motif, with six N -acetylgalactosamines, and no sialic acid. The only glycan microheterogeneity seems to reside in the number of l -fucose residues (one to three). The lack of the C-terminal cluster of basic residues, and the presence of nonsialylated glycans, result in a very low net charge of TrIFN-, molecule. However, the 17-residue truncation does not affect the antiproliferative activity of TrIFN-, on different cells, among which is a porcine uterine epithelial cell line. It is suggested that these specific properties might confer on TrIFN-, a particular ability to invade the uterine mucosa and exert biological functions beyond the endometrial epithelium. [source] Observations on the detection of b- and y-type ions in the collisionally activated decomposition spectra of protonated peptidesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 10 2009King Wai Lau Tandem mass spectrometric data from peptides are routinely used in an unsupervised manner to infer product ion sequence and hence the identity of their parent protein. However, significant variability in relative signal intensity of product ions within peptide tandem mass spectra is commonly observed. Furthermore, instrument-specific patterns of fragmentation are observed, even where a common mechanism of ion heating is responsible for generation of the product ions. This information is currently not fully exploited within database searching strategies; this motivated the present study to examine a large dataset of tandem mass spectra derived from multiple instrumental platforms. Here, we report marked global differences in the product ion spectra of protonated tryptic peptides generated from two of the most common proteomic platforms, namely tandem quadrupole-time-of-flight and quadrupole ion trap instruments. Specifically, quadrupole-time-of-flight tandem mass spectra show a significant under-representation of N-terminal b-type fragments in comparison to quadrupole ion trap product ion spectra. Energy-resolved mass spectrometry experiments conducted upon test tryptic peptides clarify this disparity; b-type ions are significantly less stable than their y-type N-terminal counterparts, which contain strongly basic residues. Secondary fragmentation processes which occur within the tandem quadrupole-time-of-flight device account for the observed differences, whereas this secondary product ion generation does not occur to a significant extent from resonant excitation performed within the quadrupole ion trap. We suggest that incorporation of this stability information in database searching strategies has the potential to significantly improve the veracity of peptide ion identifications as made by conventional database searching strategies. Copyright © 2009 John Wiley & Sons, Ltd. [source] Negative ion dissociation of peptides containing hydroxyl side chainsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 2 2008Dan Pu The dissociation of deprotonated peptides containing hydroxyl side chains was studied by electrospray ionization coupled with Fourier transform ion cyclotron resonance (ESI-FTICR) via sustained off-resonance irradiation collision induced dissociation (SORI-CID). Dissociation under post-source decay (PSD) conditions was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). This work included hexapeptides with one residue of serine, threonine, or tyrosine and five inert alanine residues. During SORI-CID and PSD, dissociation of [M,H], yielded c- and y-ions. Side-chain losses of formaldehyde (HCHO) from serine-containing peptides, acetaldehyde (CH3CHO) from threonine-containing peptides, and 4-methylene-2,5-cycohexadienone (C7H6O) from tyrosine-containing peptides were generally observed in the negative ion PSD and SORI-CID spectra. Side-chain loss occurs much less from tyrosine-containing peptides than from serine- and threonine-containing peptides. This is probably due to the bulky side chain of tyrosine, resulting in steric hindrance and poor geometry for dissociation reactions. Additionally, a selective cleavage leading to the elimination of the C-terminal residue from [M,H], was observed from the peptides with serine and threonine at the C-terminus. This cleavage does not occur in the dissociation of peptides with an amide group at the C-terminus or peptides with neutral or basic residues at the C-terminus. It also does not occur with tyrosine at the C-terminus. Both the C-terminal carboxylic acid group and the hydroxyl side chain of the C-terminal residue must play important roles in the mechanism of C-terminal residue loss. A mechanism involving both the C-terminal carboxylic acid group and a hydroxyl side chain of serine and threonine is proposed. Copyright © 2007 John Wiley & Sons, Ltd. [source] Structures of potent selective peptide mimetics bound to carboxypeptidase BACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2008Marc Adler This article reports the crystal structures of inhibitors of the functional form of thrombin-activatable fibrinolysis inhibitor (TAFIa). In vivo experiments indicate that selective inhibitors of TAFIa would be useful in the treatment of heart attacks. Since TAFIa rapidly degrades in solution, the homologous protein porcine pancreatic carboxypeptidase B (pp-CpB) was used in these crystallography studies. Both TAFIa and pp-CpB are zinc-based exopeptidases that are specific for basic residues. The final development candidate, BX 528, is a potent inhibitor of TAFIa (2,nM) and has almost no measurable effect on the major selectivity target, carboxypeptidase N. BX 528 was designed to mimic the tripeptide Phe-Val-Lys. A sulfonamide replaces the Phe-Val amide bond and a phosphinate connects the Val and Lys groups. The phosphinate also chelates the active-site zinc. The electrostatic interactions with the protein mimic those of the natural substrate. The primary amine in BX 528 forms a salt bridge to Asp255 at the base of the S1, pocket. The carboxylic acid interacts with Arg145 and the sulfonamide is hydrogen bonded to Arg71. Isopropyl and phenyl groups replace the side chains of Val and Phe, respectively. A series of structures are presented here that illustrate the evolution of BX 528 from thiol-based inhibitors that mimic a free C-terminal arginine. The first step in development was the replacement of the thiol with a phosphinate. This caused a precipitous drop in binding affinity. Potency was reclaimed by extending the inhibitors into the downstream binding sites for the natural substrate. [source] The Interaction of Highly Helical Structural Mutants with the NOP Receptor Discloses the Role of the Address Domain of Nociceptin/Orphanin FQCHEMISTRY - A EUROPEAN JOURNAL, Issue 7 2005Teodorico Tancredi Dr. Abstract Nociceptin is a heptadecapeptide whose sequence is similar to that of Dynorphin A, sharing a message domain characterized by two glycines and two aromatic residues, and a highly basic C-terminal address domain but, in spite of these similarities, displays no opioid activity. Establishing the relative importance of the message and address domains of nociceptin has so far been hampered by its extreme conformational flexibility. Here we show that mutants of this peptide, designed to increase the helical content in the address domain, can be employed to explain the mode of interaction with the NOP receptor. Nociceptin analogues in which Ala residues are substituted with aminoisobutyric acid (Aib) show a substantial increment of activity in their interaction with the NOP receptor. The increment of biological activity was attributed to the well-documented ability of Aib to induce helicity. Here we have verified this working hypothesis by a conformational investigation extended to new analogues in which the role of Aib is taken up by Leu. The NMR conformational analysis confirms that all Ala/Aib peptides as well as [Leu7,11]-N/OFQ-amide and [Leu11,15]-N/OFQ-amide mutants (N/OFQ=nociceptin/orphanin FQ) have comparable helix content in helix-promoting media. We show that the helical address domain of nociceptin can place key basic residues at an optimal distance from complementary acidic groups of the EL2 loop of the receptor. Our structural data are used to rationalize pharmacological data which show that although [Leu11,15]-N/OFQ-amide has an activity comparable to those of Ala/Aib peptides, [Leu7,11]-N/OFQ-amide is less active than N/OFQ-amide. We hypothesize that bulky residues cannot be hosted in or near the hinge region (Thr5 -Gly6 -Ala7) without severe steric clash with the receptor. This hypothesis is also consistent with previous data on this hinge region obtained by systematic substitution of Thr, Gly, and Ala with Pro. [source] | ||||||||||