CLASSIFICATION OF AMINO-ACID SPIN SYSTEMS USING PFG HCC(CO)NH-TOCSY WITH CONSTANT-TIME ALIPHATIC C-13 FREQUENCY LABELING

We have developed a useful strategy for identifying amino acid spin sy stems and side-chain carbon resonance assignments in small N-15-,C-13- enrichcd proteins. Multidimensional constant-time pulsed field gradien t (PFG) HCC(CO)NH-TOCSY experiments provide side-chain resonance frequ ency information and establish connectivities between sequential amino acid spin systems. In PFG HCC(CO)NH-TOCSY experiments recorded with a properly tuned constant-time period for frequency labeling of aliphat ic C-13 resonances, phases of cross peaks provide information that is useful for identifying spin system types. When combined with C-13 chem ical shift information, these patterns allow identification of the fol lowing spin system types: Gly, Ala, Thr, Val, Leu, Ile, Lys, Arg, Pro, long-type (i.e., Gln, Glu and Met), Ser, and AMX-type (i.e., Asp, Asn , Cys, His, Phe, Trp and Tyr).