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Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin.

ABSTRACT: Post-translational modifications are ubiquitous in the eukaryotic proteome. However, these modifications are rarely incorporated in NMR studies of eukaryotic proteins, which are typically produced through recombinant expression in E. coli. Melittin is the primary peptide in honey bee venom. Its native C-terminal amide significantly affects its equilibrium structure and dynamics in solution and is thus a prerequisite for studying its native structure and function. Here, we present a method for producing triply isotopically labeled (²H, ¹³C, and ¹⁵N) native melittin through recombinant expression followed by chemical amidation. We then show that structural models produced with AlphaFold-Multimer are in even better agreement with experimental residual dipolar couplings than the 2.0 Å resolution X-ray crystal structure for residues G3-K23.

SUBMITTER: Gelenter MD

PROVIDER: S-EPMC9951214 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin.

Gelenter Martin D MD Bax Ad A

Journal of the American Chemical Society 20230208

Post-translational modifications are ubiquitous in the eukaryotic proteome. However, these modifications are rarely incorporated in NMR studies of eukaryotic proteins, which are typically produced through recombinant expression in <i>E. coli</i>. Melittin is the primary peptide in honey bee venom. Its native C-terminal amide significantly affects its equilibrium structure and dynamics in solution and is thus a prerequisite for studying its native structure and function. Here, we present a method ...[more]

PMID: 36753641

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Project description:Optimal accuracy and precision in small-molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time-consuming, and many labeled compounds are not available in pure form. We used a single-prototype uniformly labeled [U-(13)C]compound to generate [U-(13)C]-labeled volatile standards for use in subsequent experimental profiling studies. [U-(13)C]-α-Linolenic acid (18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-(13)C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography/time-of-flight mass spectrometry (HS-SPME-GC/TOF-MS) by comparison of spectra with unlabeled volatiles. Labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-(13)C]-labeled standards, limits of detection comparable to or better than those of previous HS-SPME reports were achieved, 0.010-1.04 ng/g. The performance of the [U-(13)C]-labeled volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60 °C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-(13)C]-labeled oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-(13)C]-labeled standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-(13)C]-labeled sugars and amino acids, for profiling studies should be feasible and can dramatically improve quantitative results compared to use of a single standard.

Dataset Information

Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin.

Publications

Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin.

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