Cystine peptides: the intramolecular antiparallel beta-sheet conformation of a 20-membered cyclic peptide disulfide

A 20-membered cyclic peptide disulfide has been synthesized as a conformational model for disulfide loops of limited ring size. ¹H-nmr studies at 270 MHz establish the presence of three intramolecular hydrogen bonds involving the Leu, Val, and methylamide NH groups in CDCl₃. Evidence for peptide aggregation in CDCl₃ is also presented. A structural transition involving loosening of the hydrogen bond formed by the Val NH group is observed upon the measured addition of (CD₃)₂SO to CDCl₃. Hydrogen-bonding studies, together with unusually low field positions of the Cys(1) and Cys(6) C^αH resonances and high J values provide support for an intramolecular antiparallel β-sheet conformation, facilitated by a chain reversal at the Aib-Ala segment. Extensive nuclear Overhauser effect studies provide compelling evidence for the proposed conformation and also establish a type I′ β-turn at the Aib-Ala residues, the site of the chain reversal.     

Synthetic peptide models for the redox-active disulfide loop of glutaredoxin. Conformational studies

Two cyclic peptide disulfides (Sequence: see text). (X = L-Tyr or L-Phe) have been synthesized as models for the 14-membered redox-active disulfide loop of glutaredoxin. 1H NMR studies at 270 MHz in chloroform solutions establish a type I beta-turn conformation for the Pro-X segment in both peptides, stabilized by a 4----1 hydrogen bond between the Cys(1) CO and Cys(4) NH groups. Nuclear Overhauser effects establish that the aromatic ring in the X = Phe peptide is oriented over the central peptide unit. In dimethyl sulfoxide solutions two conformational species are observed in slow exchange on the NMR time scale, for both peptides. These are assigned to type I and type II beta-turn structures with -Pro-Tyr(Phe)- as the corner residues. The structural assignments are based on correlation of NMR parameters with model 14-membered cyclic cystine peptides with Pro-X spacers. Circular dichroism studies based on the -S-S- n-omega* transition suggest a structural change in the disulfide bridge with changing solvent polarity, establishing conformational coupling between the peptide backbone and the disulfide linkage in these systems.     

Conformations and mitochondrial uncoupling activity of synthetic emerimicin fragments

Several amino terminal fragments of the emerimicins (Ac-Phe¹-Aib²-Aib³-Aib⁴-Val⁵-Gly⁶-Leu⁷-Aib⁸-Aib⁹-Hyp¹⁰-Gln¹¹-D-Iva¹²-Hyp¹³-Ala/Aib¹⁴-Phol¹⁵) ranging in length from five to ten residues have been synthesized. Nuclear magnetic resonance studies have been carried out on the 1-5, 6-10, 1-6, 1-7, 1-8, 1-9, and 1-10 fragments. The number of solvent-shielded NH groups in CDCl₃ solutions for 1-5, 1-6, 1-7, 1-8, 1-9, and 1-10 indicate that 3₁₀-helical structures are favored in this solvent. In (CD₃)₂SO, an additional NH group, assigned to Aib(3) NH is solvent exposed in the fragments longer than six residues, suggesting partial unfolding of the N-terminal β-turn or transition to an α-helical conformation. The data for fragment 6-10 are consistent with a conformation having a single Leu-Aib β-turn. Infrared studies suggest an increase in the number of intramolecular hydrogen bonds with increasing peptide chain length. Appreciable mitochondrial uncoupling activity is observed for peptides with a chain length of at least seven residues. The order of efficiencies of the fragments is 1-7 < 1-8 ～ 1-10 < 1-9, with the decapeptide exhibiting anomalously low uncoupling activity.     

A circular dichroism study of (+) gossypol binding to proteins

The circular dichroism bands of (+) gossypol in the spectral region 300-400 nm have been shown to be sensitive to interactions with proteins. Using CD spectroscopy, gossypol has been shown to interact with lactate dehydrogenase, malate dehydrogenase, alkaline phosphatase, lysozyme, protamine and poly-L-lysine. Binding to proteins generally results in a pronounced red shift of the long wavelength CD band (approximately 380-430 nm) accompanied by a reduction in ellipticity. The changes in spectral parameters of the 1Lb binaphthyl transition may reflect a distortion from a nearly perpendicular gossypol conformation, on binding to proteins.     

Modeling,Synthesis and Characterization of Phospho–Penta–Peptide derived from PKA RII Sub Unit: A Candidate Substrate for CaN Assay

International Journal of Peptide Research and Therapeutics - Calcineurin (CaN) plays an important role in many pathological conditions like cancer, metabolic aberrations and neurodegenerative...     

Enhanced phosphorylation of AMPK by lutein and oxidised lutein that lead to mitochondrial biogenesis in hyperglycemic HepG2 cells

The stimulation of adenosine monophosphate-activated protein kinase (AMPK) is a prime target to decrease the hyperglycemic condition, hence it is a lutein (L) and oxidised lutein (OXL) is a target molecule for the treatment of type II diabetes. In the current study, a plausible interaction of L and OXL with AMPK was investigated by molecular docking. In addition, the effect of L and OXL for the activation of AMPK that triggers the downstream regulator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), TFAM expression, mitochondrial DNA (mtDNA), mitochondrial biogenesis and superoxide dismutase 2 (SOD2) in high glucose treated HepG2 cells were investigated by quantitative polymerase chain reaction and Western blot analysis. Molecular docking reveals higher binding affinity of L (ΔG = −6.3 kcal/mol) and OXL (ΔG = −15.5 kcal/mol) with AMPK, compared with metformin (ΔG = −5.0 kcal/mol). The phosphorylation of AMPK increased by 1.3- and 1.5-fold with L and OXL treatment, respectively, in high glucose induced HepG2 cells. The activation of PGC-1α is significant (P < 0.05) in OXL group than L. Similarly, TFAM expression is increased with L and OXL compared with the high glucose group. Further increase in SOD2 and mtDNA, confirms the efficacy of L and OXL in restoring the mitochondrial biogenesis in high glucose induced cells through AMPK, PGC-1α, and TFAM.     

Alamethicin and synthetic peptide fragments as uncouplers of mitochondrial oxidative phosphorylation. Effect of chain length and change

Alamethicin, its derivatives and some synthetic fragments have been shown to be uncouplers of oxidative phosphorylation in rat liver mitochondria. A minimum peptide chain length of 13 residues is necessary for this activity. Peptide esters are more efficient uncouplers than the corresponding peptide acids. Esterification of the Glu(18) γ-COOH group in alamethicin does not diminish uncoupling activity. The structural requirements for uncoupling activity parallel those determined for ionophoretic action in small, unilamellar liposomes.     

Application of HPLC to study the kinetics of a branched bi-enzyme system consisting of hypoxanthine-guanine phosphoribosyltransferase and xanthine oxidase--an important biochemical system to evaluate the efficiency of the anticancer drug 6-mercaptopurine in ALL cell line

The thiopurine antimetabolite 6-mercaptopurine (6MP) is an important chemotherapeutic drug in the conventional treatment of childhood acute lymphoblastic leukemia (ALL). 6MP is mainly catabolized by both hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine oxidase (XOD) to form thioinosinic monophosphate (TIMP) (therapeutically active metabolite) and 6-thiouric acid (6TUA) (inactive metabolite), respectively. The activity of both the enzymes varies among ALL patients governing the active and the inactive metabolite profile within the immature lymphocytes. Therefore, an attempt was made to study the kinetic nature of the branched bi-enzyme system acting on 6MP and to quantitate TIMP and 6TUA formed when the two enzymes are present in equal and variable ratios. The quantification of the branched kinetics using spectrophotometric method presents problem due to the closely apposed lambda(max) of the substrates and products. Hence, employing an HPLC method, the quantification of the products was done with the progress of time. The limit of quantification (LOQ) of substrate was found to be 10nM and for products as 50 nM. The limit of detection (LOD) was found to be 1 nM for the substrate and the products. The method exhibited linearity in the range of 0.01-100 microM for 6MP and 0.05-100 microM for both 6TUA and TIMP. The amount of TIMP formed was higher than that of 6TUA in the bi-enzyme system when both the enzymes were present in equivalent enzymatic ratio. It was further found that enzymatic ratios play an important role in determining the amounts of TIMP and 6TUA. This method was further validated using actively growing T-ALL cell line (Jurkat) to study the branched kinetics, wherein it was observed that treatment of 50 microM 6MP led to the generation of 12 microM TIMP and 0.8 microM 6TUA in 6 h at 37 degrees C.     

Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation

Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three tryptic fragments yield two nine residue sequences (FVQDFLDGK and EPQLGDRVR ) and an eleven residue sequence (DQESTGALAFK ). Database analysis using peptides and were consistent with the sequence of PDI and peptide appears to be derived from a co-migrating protein. In identifying proteins based on the characterization of short peptide sequences the question arises about the reliability of identification using peptide fragments. Here we have also demonstrated the minimum length of peptide fragment necessary for unambiguous protein identification using fragments obtained from the experimentally derived sequences. Sequences of length > or =7 residues provide unambiguous identification in conjunction with protein molecular mass as a filter. The length of sequence necessary for unambiguous protein identification is also established using randomly chosen tryptic fragments from a standard dataset of proteins. The results are of significance in the identification of proteins from organisms with unsequenced genomes.