title: 2D IR cross peaks reveal hydrogen-deuterium exchange with single residue specificity.
abstract: A form of chemical exchange, hydrogen-deuterium exchange (HDX), has long been used as a method for studying the secondary and tertiary structure of peptides and proteins using mass spectrometry and NMR spectroscopy. Using two-dimensional infrared (2D IR) spectroscopy, we resolve cross peaks between the amide II band and a (13)C(18)O isotope-labeled amide I band, which we show measures HDX with site-specific resolution. By rapidly scanning 2D IR spectra using mid-IR pulse shaping, we monitor the kinetics of HDX exchange on-the-fly. For the antimicrobial peptide ovispirin bound to membrane bilayers, we find that the amide II peak decays with a biexponential with rate constants of 0.54 ± 0.02 and 0.12 ± 0.01 min(-1), which is a measure of the overall HDX in the peptide. The cross peaks between Ile-10-labeled ovispirin and the amide II mode, which specifically monitor HDX kinetics at Ile-10, decay with a single rate constant of 0.36 ± 0.1 min(-1). Comparing this exchange rate to theoretically determined exchange rates of Ile-10 for ovispirin in a solution random coil configuration, the exchange rate at Ile-10 is at least 100 times slower, consistent with the known α-helix structure of ovispirin in bilayers. Because backbone isotope labels produce only a very small shift of the amide II band, site-specific HDX cannot be measured with FTIR spectroscopy, which is why 2D IR spectroscopy is needed for these measurements.
authors: Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53706-1396, United States.
authors: Emily B
authors: EB
authors: Dunkelberger
authors: Ann Marie
authors: AM
authors: Woys
authors: Martin T
authors: MT
authors: Zanni
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D006859:Hydrogen / Q000737:chemistry*; D007532:Isoleucine / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D010103:Oxygen Isotopes / Q000737:chemistry; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D013055:Spectrophotometry, Infrared
chemical_list: D023181:Antimicrobial Cationic Peptides; D002247:Carbon Isotopes; D008051:Lipid Bilayers; D010103:Oxygen Isotopes; D011506:Proteins; C440189:ovispirin; D007532:Isoleucine; D006859:Hydrogen; D003903:Deuterium
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