DETERMINATION OF (3)J(H-I(N),C-I(')) COUPLING-CONSTANTS IN PROTEINS WITH THE C'-FIDS METHOD

We introduce the C'-FIDS-H-1, N-15-HSQC experiment: a new method for t he determination of (3)J(H-i(N), C-i) coupling constants in proteins, yielding information about the torsional angle phi. It relies on the H -1, N-15-HSQC or HNCO experiment, two of the the most sensitive hetero nuclear correlation experiments for isotopically labeled proteins. A s et of three H-1, N-15-HSQC or HNCO spectra are recorded: a reference e xperiment in which the carbonyl spins are decoupled during t(1) and t( 2), a second experiment in which they are decoupled exclusively during t(1) and a third one in which they are coupled in t(1), as well as t( 2). The last experiment yields an E.COSY-type pattern from which the ( 2)J(HNiN, C-i-1) and (1)J(N-i, C-i-1) coupling constants can be extrac ted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first expe riment) convoluted with the (2)J(H-i(N), C-i-1) coupling, the (3)J(HN: CI) coupling can be found in a one-parameter fitting procedure. The me thod is demonstrated for the protein rhodniin, containing 103 amino ac ids. Systematic errors due to differential relaxation are small for (n )J(H-N, C-n) couplings in biomacromolecules of the size currently unde r NMR spectroscopic investigation.