The HN(COCA)HAHB NMR experiment for the stereospecific assignment of Hbeta-protons in non-native states of proteins.

(3)J(H(alpha),H(beta))-coupling constants deliver precious information on the population of the three favored chi(1)-rotamers in unfolded states of proteins. Here, a novel pulse sequence, tailored toward the NMR analysis of non-native states of proteins, the HN(COCA)HAHB experiment, is developed to measure (3)J(H(alpha),H(beta)). In four subsequent INEPT steps, magnetization is transferred from H(N) to H(alpha). In a COSY-like magnetization transfer step, dephasing of magnetization on H(alpha) is quantified to determine the (3)J(H(alpha),H(beta))-coupling constants. Analysis of the measured homonuclear coupling constants, together with measurement of heteronuclear (3)J(N,C(gamma))- and (3)J(C',C(gamma))-coupling constants, allows stereospecific assignment of the two diastereotopic H(beta)-protons even in unfolded states of proteins, and the derivation of populations according to a Pachler-type analysis.