Assignment of aliphatic side-chain 1HN/15N 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-chain 13C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D 15N/15N-separated NOESY, as many main-chain and side-chain 1HN/15N resonances as possible must be assigned. Traditionally, only backbone amide 1HN/15N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain 1HN/15N resonances to side-chain 13C resonances with high sensitivity: NH2-filtered 2D 1H-15N HSQC(H2N-HSQC), 3D H2N(CO)C gamma/beta and 3D H2N(COC gamma/beta)C beta/alpha for glutamine and asparagine side-chain amide groups; 2D refocused H(N epsilon/zeta)C delta/epsilon and H(N epsilon/zeta C delta/epsilon)C gamma/delta for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N epsilon)C zeta and nonrefocused H(N epsilon, eta)C zeta for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated 13C-/15N-labeled human carbonic anhydrase II (2H-HCA II). Because more than 95% of all side-chain 13C resonances in 2H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain 1HN/15N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain HN protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only 1HN-1HN NOE restraints. In these studies, the inclusion of NOE restraints to side-chain HN protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592-9593].
Farmer
B T
BT
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA.
Venters
R A
RA
D000595:Amino Acid Sequence; D000596:Amino Acids; D001120:Arginine; D001216:Asparagine; D003903:Deuterium; D005973:Glutamine; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D006859:Hydrogen; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity
D000596:Amino Acids; D006146:Guanidines; D009587:Nitrogen Isotopes; D011506:Proteins; D005973:Glutamine; D001216:Asparagine; D006859:Hydrogen; D001120:Arginine; D003903:Deuterium; D019791:Guanidine