Structure, dynamics and mapping of membrane-binding residues of micelle-bound antimicrobial peptides by natural abundance C NMR spectroscopy |
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Authors: | Guangshun Wang |
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Affiliation: | Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA |
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Abstract: | Worldwide bacterial resistance to traditional antibiotics has drawn much research attention to naturally occurring antimicrobial peptides (AMPs) owing to their potential as alternative antimicrobials. Structural studies of AMPs are essential for an in-depth understanding of their activity, mechanism of action, and in guiding peptide design. Two-dimensional solution proton NMR spectroscopy has been the major tool. In this article, we describe the applications of natural abundance 13C NMR spectroscopy that provides complementary information to 2D 1H NMR. The correlation of 13Cα secondary shifts with both 3D structure and heteronuclear 15N NOE values indicates that natural abundance carbon chemical shifts are useful probes for backbone structure and dynamics of membrane peptides. Using human LL-37-derived peptides (GF-17, KR-12, and RI-10), as well as amphibian antimicrobial and anticancer peptide aurein 1.2 and its analog LLAA, as models, we show that the cross peak intensity plots of 2D 1H-13Cα HSQC spectra versus residue number present a wave-like pattern (HSQC wave) where key hydrophobic residues of micelle-bound peptides are located in the troughs with weaker intensities, probably due to fast exchange between the free and bound forms. In all the cases, the identification of aromatic phenylalanines as a key membrane-binding residue is consistent with previous intermolecular Phe-lipid NOE observations. Furthermore, mutation of one of the key hydrophobic residues of KR-12 to Ala significantly reduced the antibacterial activity of the peptide mutants. These results illustrate that natural abundance heteronuclear-correlated NMR spectroscopy can be utilized to probe backbone structure and dynamics, and perhaps to map key membrane-binding residues of peptides in complex with micelles. 1H-13Cα HSQC wave, along with other NMR waves such as dipolar wave and chemical shift wave, offers novel insights into peptide-membrane interactions from different angles. |
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Keywords: | NMR, nuclear magnetic resonance AMPs, antimicrobial peptides APD2, the antimicrobial peptide database version 2 D8PG, dioctanoyl phosphatidylglycerol DPC, dodecylphosphocholine DQF-COSY, double-quantum filtered correlation spectroscopy DSS, 2,2-dimethyl-silapentane-5-sulfonate sodium salt HSQC, heteronuclear single-quantum coherence spectroscopy LLAA, LL-37-derived aurein 1.2 analog MIC, minimal inhibitory concentration NOE, nuclear Overhauser effect NOESY, nuclear Overhauser enhancement spectroscopy PGs, phosphatidylglycerols SDS, sodium dodecylsulfate TOCSY, total correlation spectroscopy |
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