Synthesis, conformation, and activity of human insulin-like peptide 5 (INSL5)

Insulin-like peptide 5 (INSL5) was first identified through searches of the expressed sequence tags (EST) databases. Primary sequence analysis showed it to be a prepropeptide that was predicted to be processed in vivo to yield a two-chain sequence (A and B) that contained the insulin-like disulfide cross-links. The high affinity interaction between INSL5 and the receptor RXFP4 (GPCR142) coupled with their apparent coevolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for RXFP4. Given that the primary function of the INSL5-RXFP4 pair remains unknown, an effective means of producing sufficient quantities of this peptide and its analogues is needed to systematically investigate its structural and biological properties. A combination of solid-phase peptide synthesis methods together with regioselective disulfide bond formation were used to obtain INSL5. Both chains were unusually resistant to standard synthesis protocols and required highly optimized conditions for their acquisition. In particular, the use of a strong tertiary amidine, DBU, as N(alpha)-deprotection base was required for the successful assembly of the B chain; this highlights the need to consider incomplete deprotection rather than acylation as a cause of failed synthesis. Following sequential disulfide bond formation and chain combination, the resulting synthetic INSL5, which was obtained in good overall yield, was shown to possess a similar secondary structure to human relaxin-3 (H3 relaxin). The peptide was able to inhibit cAMP activity in SK-N-MC cells that expressed the human RXFP4 receptor with a similar activity to H3 relaxin. In contrast, it had no activity on the human RXFP3 receptor. Synthetic INSL5 demonstrates equivalent activity to the recombinant-derived peptide, and will be an important tool for the determination of its biological function.     

Determination of the conformation of the human VDAC1 N-terminal peptide, a protein moiety essential for the functional properties of the pore

Mitochondrial porin or VDAC (voltage-dependent anion-selective channel) is the most abundant protein in the mitochondrial outer membrane. The structure of VDAC has been predicted to be a transmembrane beta-barrel with an alpha-helix at the N terminus. It is a matter of debate as to whether this putative alpha-helix plays a structural role as a component of the pore walls or a function in the pore activity. We have synthesised the human VDAC1 (HVDAC1) N-terminal peptide Ac-AVPPTYADLGKSARDVFTK-NH2 (Prn2-20) and determined its structure by CD and NMR spectroscopy. CD studies show that the Prn2-20 peptide exists in aqueous solvent as an unstructured peptide with no stable secondary structure. In membrane-mimetic SDS micelles or water/trifluoroethanol, however, it assumes an amphipathic alpha-helix conformation between Tyr5 and Val16, as deduced from NMR. No ordered structure was observed in dodecyl beta-maltoside. Differential scanning calorimetric measurements were carried out in order to examine the membrane affinity of the peptide. Upon interaction with the negatively charged 1,2 dipalmitoyl-sn-glycero-3-phosphoserine membrane, Prn2-20 exhibited distinctive behaviour, suggesting that electrostatics play an important role. Interaction between the peptide and artificial bilayers indicates that the peptide lies on the membrane surface. Recombinant HVDAC1 deletion mutants, devoid of seven or 19 N-terminal amino acids, were used for transfection of eukaryotic cells. Over-expression of HVDAC1 increases the number of Cos cells with depolarised mitochondria, and this effect is progressively reduced in cells transfected with HVDAC1 lacking those seven or 19 amino acids. The mitochondrial targeting of the deletion mutants is unaffected. The overall picture emerging from our experiments is that the VDAC N-terminal peptide plays a role in the proper function of this protein during apoptotic events.     

Metal Binding Properties of Fluorescent Analogues of Trichogin GA IV: A Conformational Study by Time‐Resolved Spectroscopy and Molecular Mechanics Investigations

The metal ion binding properties of two fluorescent analogues of trichogin GA IV, which is a natural undecapeptide showing significant antimicrobial activity, were studied by circular dichroism, time‐resolved optical spectroscopy, and molecular mechanics calculations. Binding of Ca^II and Gd^III to the peptides investigated was shown to promote a structural transition from highly helical conformations to folded structures characterized by formation of a loop that embedded the metal ion. Time‐resolved spectroscopy revealed that peptide dynamics is also remarkably affected by ion binding: peptide‐backbone motions slowed down to the microsecond time scale. Finally, molecular mechanics calculations emphasized the role of the central Gly5‐Gly6 motif, which allowed for the twisting of the peptide segment that gave rise to the formation of the binding cavity.     

New Antimicrobial Hexapeptides: Synthesis,Antimicrobial Activities,Cytotoxicity, and Mechanistic Studies

Synthetic antimicrobial peptides have recently emerged as promising candidates against drug‐resistant pathogens. We identified a novel hexapeptide, Orn‐D ‐Trp‐D ‐Phe‐Ile‐D ‐Phe‐His(1‐Bzl)‐NH₂, which exhibits broad‐spectrum antifungal and antibacterial activity. A lead optimization was undertaken by conducting a full amino acid scan with various proteinogenic and non‐proteinogenic amino acids depending on the hydrophobic or positive‐charge character of residues at various positions along the sequence. The hexapeptide was also cyclized to study the correlation between the linear and cyclic structures and their respective antimicrobial activities. The synthesized peptides were found to be active against the fungus Candida albicans and Gram‐positive bacteria such as methicillin‐resistant Staphylococcus aureus and methicillin‐resistant Staphylococcus epidermidis, as well as the Gram‐negative bacterium Escherichia coli; MIC values for the most potent structures were in the range of 1–5 μg mL^?1 (IC₅₀ values in the range of 0.02–2 μg mL^?1). Most of the synthesized peptides showed no cytotoxic effects in an MTT assay up to the highest test concentration of 200 μg mL^?1. A tryptophan fluorescence quenching study was performed in the presence of negatively charged and zwitterionic model membranes, mimicking bacterial and mammalian membranes, respectively. The results of the fluorescence study demonstrate that the tested peptides are selective toward bacterial over mammalian cells; this is associated with a preferential interaction between the peptides and the negatively charged phospholipids of bacterial cells.     

Asymmetric Contribution of Aromatic Interactions Stems from Spatial Positioning of the Interacting Aryl Pairs in β‐Hairpins

Isolated aromatic interactions in designed octapeptide β‐hairpin scaffolds display a near‐universal T‐shaped face‐to‐edge geometry in all positional permutations, with the exception of aryl‐Trp interactions. The heterogeneous asymmetric indole ring of Trp competes for a “shielding” face geometry, which lowers the scaffold stability in FtE aryl‐Trp pairs. Assessment of the contributions of aryl pairs (in the absence of solvent‐driven interactions) to the overall β‐hairpin structure reveals the superiority of Trp‐Phe and Trp‐Tyr contributions over the well‐established scaffold stabilization by Trp‐Trp.     

Applications of circular dichroism for structural analysis of gelatin and antimicrobial peptides

Circular dichroism (CD) is a useful technique for monitoring changes in the conformation of antimicrobial peptides or gelatin. In this study, interactions between cationic peptides and gelatin were observed without affecting the triple helical content of the gelatin, which was more strongly affected by anionic surfactant. The peptides did not adopt a secondary structure in the presence of aqueous solution or Tween 80, but a peptide secondary structure formed upon the addition of sodium dodecyl sulfate (SDS). The peptides bound to the phosphate group of lipopolysaccharide (LPS) and displayed an alpha-helical conformation while (KW)(4) adopted a folded conformation. Further, the peptides did not specifically interact with the fungal cell wall components of mannan or laminarin. Tryptophan blue shift assay indicated that these peptides interacted with SDS, LPS, and gelatin but not with Tween 80, mannan, or laminarin. The peptides also displayed antibacterial activity against P. aeruginosa without cytotoxicity against HaCaT cells at MIC, except for HPA3NT3-analog peptide. In this study, we used a CD spectroscopic method to demonstrate the feasibility of peptide characterization in numerous environments. The CD method can thus be used as a screening method of gelatin-peptide interactions for use in wound healing applications.     

Structural Alterations of Human Serum Albumin Caused by Glycative and Oxidative Stressors Revealed by Circular Dichroism Analysis

The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points.     

光谱技术研究硫堇与牛血清白蛋白的结合反应

用荧光光谱、紫外可见吸收光谱和圆二色光谱技术研究了生理条件下硫堇(TH)与牛血清白蛋白(BSA)的结合反应.结果表明,硫堇借静电力与牛血清白蛋白发生相互作用并对牛血清白蛋白的荧光以静态方式猝灭,其结合常数为1.155×105L·mol-1、结合位点数为1.835、结合距离2.865 nm.同步荧光和圆二色技术证实TH对BSA的构像有影响.