Structural and conformational study of peptides containing glycine and alanine residues by C-13 CPMAS NMR spectroscopy in solid state

The results of the structural and conformational studies carried out using C-13 CPMAS NMR technique on several glycine and alanine containing peptides in the solid state are reported. The study demonstrates the effects of variations in C-13 chemical shifts due to conformation and hydrogen bonding. The possibility of applying this technique to obtain insight into the conformational characteristics of peptides of unknown structures is discussed.

Structure and activity cyclase activated of OK-GC: A kidney receptor-guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4�75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Structure and activity of OK-GC: a kidney receptor guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Synthesis of two designed hairpin peptides on a newly developed 1,6-hexanediol diacrylate (HDODA)-crosslinked polystyrene resin

Synthesis of two designed hairpin peptides on 1,6-hexanediol diacrylate crosslinked polystyrene support using the standard solid phase methodology is described. Both the peptides are obtained in high yield and purity. The new polymeric system is an ideal support for the synthesis of hairpin peptides, which is a very difficult task by the solid phase method.

The prop-2-ynyloxy carbonyl function (POC): A new amino-protecting group removable from sulfur-containing peptides by ultrasonic irradiation with tetrathiomolybdate under mild and neutral conditions

The prop-2-ynyloxy carbonyl function (POC) which can be cleaved under mild and neutral conditions in the presence of benzyltriethylammonium tetrathiomolybdate has been developed as a new protecting group for amines. (C) 1999 Elsevier Science Ltd. All rights reserved.

Propargyloxycarbonyl (Poc) amino acid chlorides as efficient coupling reagents for the synthesis of 100% diastereopure peptides and resin bound tetrathiomolybdate as an effective deblocking agent for the Poc group

Synthesis of short peptides using propargyloxycarbonyl amino acid chlorides as effective coupling reagents and polymer supported tetrathiomolybdate as an efficient deblocking agent are reported.

The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function

In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of the second step of pre-mRNA splicing. The function and interacting factors for this protein have not been elucidated. We have performed a mutational analysis of yPrp17p to identify protein domains critical for function. A series of deletions were made throughout the region spanning the N-terminal 158 amino acids of the protein, which do not contain any identified structural motifs. The C-terminal portion (amino acids 160–455) contains a WD domain containing seven WD repeats. We determined that a minimal functional Prp17p consists of the WD domain and 40 amino acids N-terminal to it. We generated a three-dimensional model of the WD repeats in Prp17p based on the crystal structure of the [beta]-transducin WD domain. This model was used to identify potentially important amino acids for in vivo functional characterization. Through analysis of mutations in four different loops of Prp17p that lie between [beta] strands in the WD repeats, we have identified four amino acids, 235TETG238, that are critical for function. These amino acids are predicted to be surface exposed and may be involved in interactions that are important for splicing. Temperature-sensitive prp17 alleles with mutations of these four amino acids are defective for the second step of splicing and are synthetically lethal with a U5 snRNA loop I mutation, which is also required for the second step of splicing. These data reinforce the functional significance of this region within the WD domain of Prp17p in the second step of splicing.

Accessing the disallowed conformations of peptides employing amide-to-imidate modification

Selective modification of the C-terminal amide in peptides to dihydrooxazine (a novel stable imidate isostere) by intramolecular nucleophilic cyclo-O-alkylation of the corresponding N-(3-bromopropyl)amides results in constraining of the C-terminal residue in natively disallowed conformations both in crystals and in solution.

Self-Assembling Peptides: From Molecules to Nanobiomaterials

Abstract | Molecular self-assembly plays a vital role in the construction of various nanostructures using the ‘bottom-up’ approach. Peptides have been considered important bio-molecular building blocks for different nanoscale structures as they are biocompatible, biodegradable, generally non-toxic and can be attuned to environmental responses like pH, temperature, salt concentration and others. Peptide based nanostructures can offer various wonderful biological applications in tissue engineering, cell culture, regenerative medicine and drug delivery. In this review, the construction of short peptide-based different nanostructures including nanotubes, nanovesicles and nanofibers, short peptide-based nanoporous materials, short peptide-based nanofibrous hydrogels and nanovesicles for various biological applications has been discussed. Moreover, morphological transformations from one nanoscopic structure to an other type of nanostructure (e.g., nanotubes to nanovesicles) are also clearly discussed in this review.

NMRof peptides

Developments and applications of NMR spectroscopy especially with biomolecules has taken big strides over the decades. This review gives a brief overview of peptide analysis by NMR as carried out in the author’s laboratory. A brief introduction to peptide biomolecules and NMR useful parameters are discussed in the beginning. This is followed by diagnostics features observed in NMR for identification of secondary structures. It further goes on to show how a three dimensional structure could be obtained by all-important NOE and hydrogen bond information. Use of heteronuclear experiments, which could be done at natural abundance is also highlighted in getting more details of peptide structures.Applications using Solid state NMR at natural abundance in connecting peptide solution and x-ray structures is demonstrated with couple of examples.

NMR Structure Implications of Enhanced Efficacy of Obestatin Fragment Analogs

Obestatin is a more recently discovered hormone that is encoded by the ghrelin gene and produced in the stomach and gut. We report NMR analysis on synthetic Obestatin (OB23), a 23 residue peptide, along with three overlapping fragments of the same in methanol solvent as a first step towards structure activity relationship. Selective substitutions on the promising N-terminal and middle fragments of obestatin have been carried out in order to improve the efficacy and potency. In the N-terminal fragment two peptides were obtained by the replacement of Gly (8) with a-aminoisobutyric acid (Aib, U) and Phe (F5) with Cyclohexylalanine (Cha). In case of the middle fragment both Gly (3) and Gly (8) were replaced with Aib residues. The rationale being, these unusual amino acids could provide protection from immediate degradation and aid structure stabilization. Our previous studies showed that the N-terminal and the middle fragment were unstructured and hence this substitution would directly evaluate the effect of structure on the activity of these fragment analogs. Detailed NMR analysis clearly demonstrates formation of helical secondary structure in all the peptide analogues and provides justification for relative activities reported by our group previously (Nagaraj et al. 2009).

Saccharomyces cerevisiae NineTeen Complex (NTC)-associated Factor Bud31/Ycr063w Assembles on Precatalytic Spliceosomes and Improves First and Second Step Pre-mRNA Splicing Efficiency

Pre-mRNA splicing occurs in spliceosomes whose assembly and activation are critical for splice site selection and catalysis. The highly conserved NineTeen complex protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NineTeen complex-associated proteins is the nonessential protein Bud31/Ycr063w, which is also a component of the Cef1p subcomplex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors, but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed that its earliest association is with precatalytic B complex and that the interaction continues in catalytically active complexes with stably bound U2, U5, and U6 small nuclear ribonucleoproteins. In complementary experiments, wherein precatalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Cross-linking of proteins to pre-mRNAs with a site-specific 4-thio uridine residue at the -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a similar to 25-kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.