Heteronuclear gradient-enhanced NMR for the study of 20–30kDa proteins: Application to human carbonic anhydrase II
Human carbonic anhydrase (HCA) is ubiquitous in living systems with seven different mammalian isozymes (CAI to CAVII), and HCAII is one of the largest monomeric proteins currently, being studied by NMR, making it a good system on which to demonstrate the advantages of pulsed field gradients (PFG) technology. This chapter describes the utilization of 2-D and 3-D homonuclear and heteronuclear NMR experiments optimized for use with proteins and peptides including the enzyme HCA. Pulsed field gradients are added to existing NMR pulse sequences to suppress artifacts and/or to select certain coherence transfer pathways. The signal-to-noise ratio obtained is better when higher power shorter duration gradients are used, as long as the gradient times do not become too short for proper amplifier response. These results can be used directly to implement gradient versions of NH detected 13C/15N/1H 3-D experiments for protein assignments purposes resulting in data with higher sensitivity and less coherent noise using significantly shorter instrument times, when compared with their non-gradient precursors. These experiments allow for the study of proteins 30kDa and larger greatly expanding the quantity and types of systems now accessible. © 1995, Elsevier Inc.
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