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Single-molecule Study on the Interactions between Cyclic Nonribosomal Peptides and Protein Nanopore.

ABSTRACT: Nonribosomal peptides (NRPs) are a type of secondary metabolites mostly originated from microorganisms such as bacteria and fungi. Their proteolytic stability, highly selective bioactivity, and microorganism-specificity have made them an attractive source of drugs for the pharmaceutical industry. Herein, with microcystins (MCs) as a NRP model, we, for the first time, proposed a sensitive method to study the interactions between NRPs and the protein nanopore. Due to the large molecular size (~3 nm diameter) of MCs and their net negative charges, MCs failed to translocate through the α-hemolysin (α-HL) protein channel. Our results demonstrated that the biomolecular interaction of MC-α-HL protein was significantly affected by the applied potential bias. The constant blockage amplitude in the voltage-dependent studies indicated that the current modulation events were dominantly contributed to the bumping interaction between MCs and the α-HL protein under the electrophoretic force. The mean residence time of the bumping events exhibited a two-stage decrease (from 1.90 ms to 1.02 ms, and from 1.02 ms to 0.69 ms) at the threshold voltages of -70 mV and -100 mV, respectively. Using our strategy (i.e., based on their electrophoretic driven interaction with the α-HL protein pore), discrimination of different MC molecules (MC-LR, MC-RR, MC-YR and linear analog) with varied branched residues could be accomplished. This work should provide an insight in developing a rapid and effective method for the identification of cyclic NRPs as valuable biomarkers for fungal infections.

SUBMITTER: Zhou S

PROVIDER: S-EPMC8221584 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Natural and non-natural cyclic peptides are a crucial component in drug discovery programs because of their considerable pharmaceutical properties. Cyclosporin, microcystins, and nodularins are all notable pharmacologically important cyclic peptides. Because these biologically active peptides are often biosynthesized nonribosomally, they often contain nonstandard amino acids, thus increasing the complexity of the resulting tandem mass spectrometry data. In addition, because of the cyclic nature, the fragmentation patterns of many of these peptides showed much higher complexity when compared to related counterparts. Therefore, at the present time it is still difficult to annotate cyclic peptides MS/MS spectra. In this current work, an annotation program was developed for the annotation and characterization of tandem mass spectra obtained from cyclic peptides. This program, which we call MS-CPA is available as a web tool (http://lol.ucsd.edu/ms-cpa_v1/Input.py). Using this program, we have successfully annotated the sequence of representative cyclic peptides, such as seglitide, tyrothricin, desmethoxymajusculamide C, dudawalamide A, and cyclomarins, in a rapid manner and also were able to provide the first-pass structure evidence of a newly discovered natural product based on predicted sequence. This compound is not available in sufficient quantities for structural elucidation by other means such as NMR. In addition to the development of this cyclic annotation program, it was observed that some cyclic peptides fragmented in unexpected ways resulting in the scrambling of sequences. In summary, MS-CPA not only provides a platform for rapid confirmation and annotation of tandem mass spectrometry data obtained with cyclic peptides but also enables quantitative analysis of the ion intensities. This program facilitates cyclic peptide analysis, sequencing, and also acts as a useful tool to investigate the uncommon fragmentation phenomena of cyclic peptides and aids the characterization of newly discovered cyclic peptides encountered in drug discovery programs.

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Single-molecule Study on the Interactions between Cyclic Nonribosomal Peptides and Protein Nanopore.

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