Methyl side-chain dynamics in proteins using selective enrichment with a single isotopomer.
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.
Chaykovski
Michael M
MM
Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, LS2 9JT UK.
Bae
Lynnette C
LC
Cheng
Minn-Chang
MC
Murray
Jenny H
JH
Tortolani
Kenneth E
KE
Zhang
Rui
R
Seshadri
Kothandaraman
K
Findlay
John H B C
JH
Hsieh
Shih-Yang
SY
Kalverda
Arnout P
AP
Homans
Steve W
SW
Brown
Jonathan Miles
JM
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
D002247:Carbon Isotopes; D011506:Proteins; C035737:major urinary proteins; D003903:Deuterium; D014633:Valine