RESUMO
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.
Assuntos
Proteínas/química , Animais , Isótopos de Carbono , Deutério , Camundongos , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Valina/químicaRESUMO
The implementation of [13Calpha,13C',15N,2Halpha] labelled amino acids into proteins allows the acquisition of high resolution triple resonance experiments. We present for the first time resonance assignments facilitated by this new labelling strategy. The absence of 1JCalpha,Cbeta couplings enables us to measure 1JCalpha,C' scalar and 1DCalpha,C' residual dipolar coupling constants using modified HNCA experiments which do not suffer from sensitivity losses characteristic for 13C constant time experiments.
