Sequence-specific model for predicting peptide collision cross-section values in proteomic ion mobility spectrometry

ABSTRACTThe contribution of peptide amino-acid sequence to collision cross-section values (CCS) has been investigated using a dataset of ∼134,000 peptides of four different charge states (1+ to 4+). The migration data was acquired using a two-dimensional LC/trapped ion mobility spectrometry/quadrupole/time-of-flight MS analysis of HeLa cell digests created using 7 different proteases and was converted to CCS values. Following the previously reported modeling approaches using intrinsic size parameters (ISP), we extended this methodology to encode the position of individual residues within a peptide sequence. A generalized prediction model was built by dividing the dataset into 8 groups (four charges for both tryptic/non-tryptic peptides). Position dependent ISPs were independently optimized for the eight subsets of peptides, resulting in prediction accuracy of ∼0.981 for the entire population of peptides. We find that ion mobility is strongly affected by the peptide’s ability to solvate the positively charged sites. Internal positioning of polar residues and proline leads to decreased CCS values as they improve charge solvation; conversely, this ability decreases with increasing peptide charge due to electrostatic repulsion. Furthermore, higher helical propensity and peptide hydrophobicity result in preferential formation of extended structures with higher than predicted CCS values. Finally, acidic/basic residues exhibit position dependent ISP behaviour consistent with electrostatic interaction with the peptide macro-dipole, which affects the peptide helicity.

Screening and Rational Design of Hepatitis C Virus Entry Inhibitory Peptides Derived from GB Virus A NS5A

ABSTRACTChronic infection by hepatitis C virus (HCV) is a cause of the global burden of liver diseases. HCV entry into hepatocytes is a complicated and multistep process that represents a promising target for antiviral intervention. The recently reported amphipathic α-helical virucidal peptide (C5A) from the HCV NS5A protein suggests a new category of antiviral drug candidates. In this study, to identify C5A-like HCV inhibitors, synthetic peptides derived from the C5A-corresponding NS5 protein region of selectedFlaviviridaeviruses were evaluated for their anti-HCV activities. A peptide from GB virus A (GBV-A), but not other flaviviruses, demonstrated an inhibitory effect on HCV infection. Through a series of sequence optimizations and modifications of the peptide helicity and hydrophobicity, we obtained a peptide designated GBVA10-9 with highly potent anti-HCV activity. GBVA10-9 suppressed infection with both cell culture-derived and pseudotyped HCVin vitro, and the 50% cell culture inhibitory concentration ranged from 20 nM to 160 nM, depending on the genotypic origin of the envelope proteins. GBVA10-9 had no detectable effects on either HCV attachment to Huh7.5.1 cells or viral RNA replication. No virucidal activity was found with GBVA10-9, suggesting an action mechanism distinct from that of C5A. The inhibitory effect of GBVA10-9 appeared to occur at the postbinding step during viral entry. Taken together, the results with GBVA10-9 demonstrated a potent activity for blocking HCV entry that might be used in combination with other antivirals directly targeting virus-encoded enzymes. Furthermore, GBVA10-9 also provides a novel tool to dissect the detailed mechanisms of HCV entry.

Role of Peptide Hydrophobicity in the Mechanism of Action of α-Helical Antimicrobial Peptides

ABSTRACT In the present study, the 26-residue amphipathic α-helical antimicrobial peptide V13KL (Y. Chen et al., J. Biol. Chem. 2005, 280:12316-12329, 2005) was used as the framework to study the effects of peptide hydrophobicity on the mechanism of action of antimicrobial peptides. Hydrophobicity was systematically decreased or increased by replacing leucine residues with less hydrophobic alanine residues or replacing alanine residues with more hydrophobic leucine residues on the nonpolar face of the helix, respectively. Hydrophobicity of the nonpolar face of the amphipathic helix was demonstrated to correlate with peptide helicity (measured by circular dichroism spectroscopy) and self-associating ability (measured by reversed-phase high-performance liquid chromatography temperature profiling) in aqueous environments. Higher hydrophobicity was correlated with stronger hemolytic activity. In contrast, there was an optimum hydrophobicity window in which high antimicrobial activity could be obtained. Decreased or increased hydrophobicity beyond this window dramatically decreased antimicrobial activity. The decreased antimicrobial activity at high peptide hydrophobicity can be explained by the strong peptide self-association which prevents the peptide from passing through the cell wall in prokaryotic cells, whereas increased peptide self-association had no effect on peptide access to eukaryotic membranes.