CHARACTERIZING THE USE OF PERDEUTERATION IN NMR-STUDIES OF LARGE PROTEINS C-13, N-15 AND H-1 ASSIGNMENTS OF HUMAN CARBONIC-ANHYDRASE-II

Perdeuteration of all non-exchangeable proton sites can significantly increase the size of proteins and protein complexes for which NMR reso nance assignments and structural studies are possible. Backbone H-1, N -15, (CO)-C-13, C-13(alpha) and C-13(beta) chemical shifts and aliphat ic side-chain C-13 and H-1(N)/N-15 chemical shifts for human carbonic anhydrase II (HCA II), a 259 residue 29 kDa metalloenzyme, have been d etermined using a strategy based on 2D, 3D and 4D heteronuclear NMR ex periments, and on perdeuterated C-13/N-15-labeled protein. To date, HC A II is one of the largest monomeric proteins studied in detail by hig h-resolution NMR. Of the backbone resonances, 85% have been assigned u sing fully protonated N-15 and C-3/N-15-labeled protein in conjunction with established procedures based on now standard 2D and 3D NMR exper iments. HCA II has been perdeuterated both to complete the backbone re sonance assignment and to assign the aliphatic side-chain C-13 and H-1 (N)/N-15 resonances. The incorporation of H-2 into HCA II dramatically decreases the rate of C-13 and (HNT2)-H-1 relaxation. This, in turn, increases the sensitivity of several key H-1/C-13/N-15 triple-resonanc e correlation experiments. Many otherwise marginal heteronuclear 3D an d 4D correlation experiments, which are important to the assignment st rategy detailed herein, can now be executed successfully on HCA II. Fu rther analysis suggests that, from the perspective of sensitivity, per deuteration should allow other proteins with rotational correlation ti mes significantly longer than HCA II (tau(c) = 11.4 ns) to be studied successfully with these experiments. Two different protocols have been used to characterize the secondary structure of HCA II from backbone chemical-shift data. Secondary structural elements determined in this manner compare favorably with those elements determined from a consens us analysis of the HCA II crystal structure. Finally, having outlined a general strategy for assigning backbone and side-chain resonances in a perdeuterated large protein, we propose a strategy whereby this inf ormation can be used to glean more detailed structural information fro m the partially or fully protonated protein equivalent. (C) 1996 Acade mic Press Limited