ASSIGNMENT OF ALIPHATIC SIDE-CHAIN H-1(N) N-15 RESONANCES IN PERDEUTERATED PROTEINS/

The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-cha in C-13 assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the max imum information from a 4D N-15/N-15-separated NOESY, as many main-cha in and side-chain H-1(N)/N-15 resonances as possible must be assigned. Traditionally, only backbone amide H-1(N)/N-15 resonances are assigne d by correlation experiments, whereas slowly exchanging side-chain ami de, amino, and guanidino protons are assigned by NOEs to side-chain al iphatic protons. In a perdeuterated protein, however, there is a minim al number of such protons. We have therefore developed several gradien t-enhanced and sensitivity-enhanced pulse sequences, containing water- flipback pulses, to provide through-bond correlations of the aliphatic sidechain H-1(N)/N-15 resonances to side-chain C-13 resonances with h igh sensitivity: NH2-filtered 2D H-1-N-15 HSQC (H2N-HSQC), 3D H2N(CO)C -gamma/beta and 3D H2N(COCgamma/beta)C-beta/alpha for glutamine and as paragine side-chain amide groups; 2D refocused H(N-epsilon/zeta)C-delt a/epsilon and H(Nepsilon/zetaCdelta/epsilon)C-gamma/delta for arginine side-chain amino groups and non-refocused versions for lysine side-ch ain amino groups; and 2D refocused H(N-epsilon)C-zeta and nonrefocused H(N-epsilon eta)C-zeta for arginine side-chain guanidino groups. Thes e pulse sequences have been applied to perdeuterated C-13-/N-15-labele d human carbonic anhydrase II (H-2-HCA II). Because more than 95% of a ll side-chain C-13 resonances in H-2-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain H-1 (N)/N-15 resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain H, prot ons has been demonstrated by recent studies in which the calculation o f protein global folds was simulated using only H-1(N)-H-1(N), NOE res traints. In these studies, the inclusion of NOE restraints to side-cha in HN protons significantly improved the quality of the global fold th at could be determined for a perdeuterated protein [R.A. Venters et al . (1995) J. Am. Chem. Soc., 117, 9592-9593].