A novel approach for the sequential backbone assignment of larger proteins: selective intra-HNCA and DQ-HNCA.

Abstract

Sequential assignment of backbone resonances in larger proteins can be achieved by recording two or more complementary triple-resonance NMR spectra of deuterated proteins. For such proteins, higher fields and experiments based on the TROSY method provide the needed resolution and sensitivity. However, increasingly rapid carbonyl relaxation at the high magnetic field strengths required by TROSY techniques renders assignment strategies that rely on sequential HN(CO)CA-type experiments much less efficient for proteins >40 kDa. Here we present two complementary new experiments, which allow backbone assignments with good sensitivity for larger deuterated proteins. A 3D intra-HNCA experiment provides uniquely the intraresidue connection, while a 3D DQ-HNCA experiment, which detects a (13)C(alpha)(i)()(13)C(alpha)(i-1)() double-quantum (DQ) coherence, contains the sequential information. The experiments work well at high magnetic fields, and their utility is demonstrated on a protein with a correlation time of 28 ns ( approximately 60 kDa). For larger proteins the sensitivity is predicted through simulations which suggest that the approach should work for proteins with correlation times >50 ns.