title: Methyl side-chain dynamics in proteins using selective enrichment with a single isotopomer.
abstract: 13C relaxation studies on side-chain methyl groups in proteins typically involve measurements on (13)CHD(2) isotopomers, where the (13)C relaxation mechanism is particularly straightforward in the presence of a single proton. While such isotopomers can be obtained in proteins overexpressed in bacteria by use of (13)C enriched and fractionally deuterated media, invariably all possible (2)H isotopomers are obtained. This results in a loss of both resolution and sensitivity, which becomes particularly severe for larger proteins. We describe an approach that overcomes this problem by chemical synthesis of amino acids containing a pure (13)CHD(2) isotopomer. We illustrate the benefits of this approach in (13)C side-chain relaxation measurements on the mouse major urinary protein selectively enriched with [gamma(1),gamma(2)-(13)C(2),alpha,beta,gamma(1),gamma(1),gamma(2),gamma(2)-(2)H(6)] valine. Relaxation measurements in the absence and presence of pyrazine-derived ligands suggest that valine side-chain dynamics do not contribute significantly to binding entropy.
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, LS2 9JT UK.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Chaykovski
authors.forename: Lynnette C
authors.initials: LC
authors.lastname: Bae
authors.forename: Minn-Chang
authors.initials: MC
authors.lastname: Cheng
authors.forename: Jenny H
authors.initials: JH
authors.lastname: Murray
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Tortolani
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Kothandaraman
authors.initials: K
authors.lastname: Seshadri
authors.forename: John H B C
authors.initials: JH
authors.lastname: Findlay
authors.forename: Shih-Yang
authors.initials: SY
authors.lastname: Hsieh
authors.forename: Arnout P
authors.initials: AP
authors.lastname: Kalverda
authors.forename: Steve W
authors.initials: SW
authors.lastname: Homans
authors.forename: Jonathan Miles
authors.initials: JM
authors.lastname: Brown
mesh_terms: D000818:Animals; D002247:Carbon Isotopes; D003903:Deuterium; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D014633:Valine / Q000737:chemistry
chemical_list: D002247:Carbon Isotopes; D011506:Proteins; C035737:major urinary proteins; D003903:Deuterium; D014633:Valine