New methylene specific experiments for the measurement of scalar spin-spin coupling constants between protons attached to 13C.

New two- and three-dimensional NMR methods are proposed for the measurement of 3J(H, H) coupling constants between two adjacent methylene moieties. The new experiment, which is based on a combination of the E.COSY principle and double/zero quantum heteronuclear spectroscopy, has been applied to diaceton-glucose and to the protein rhodniin. The coupling constants of CH-CH2 groups have been compared with those obtained from a HCCH-E.COSY experiment to check the reliability of the results. An analysis of the coupling constants derived by comparison between experimental and simulated spectra is presented. Simulations were done with the program wtest considering fully correlated dipolar relaxation. Side-chain conformations in amino acids with adjacent methylene groups can be determined by the new experiment.

Determination of (3)J(H (infi) (supN) ,C (infi) (sup') ) coupling constants in proteins with the C'-FIDS method.

We introduce the C'-FIDS-(1)H,(15)N-HSQC experiment, a new method for the determination of (3)J(H (infi) (supN) ,C (infi) (sup') ) coupling constants in proteins, yielding information about the torsional angle ϕ. It relies on the (1)H,(15)N-HSQC or HNCO experiment, two of the the most sensitive heteronuclear correlation experiments for isotopically labeled proteins. A set of three (1)H,(15)N-HSQC or HNCO spectra are recorded: a reference experiment 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(H (infi) (supN) ,C (infi-1) (sup') ) and (1)J(N(i),C (infi-1) (sup') ) coupling constants can be extracted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first experiment) convoluted with the (2)J(H (infi) (supN) ,C (infi-1) (sup') ) coupling, the (3)J(H (infi) (supN) ,C (infi) (sup') ) coupling can be found in a one-parameter fitting procedure. The method is demonstrated for the protein rhodniin, containing 103 amino acids. Systematic errors due to differential relaxation are small for (n)J(H(N),C') couplings in biomacromolecules of the size currently under NMR spectroscopic investigation.