High-throughput backbone resonance assignment of small 13C,15N-labeled proteins by a triple-resonance experiment with four sequential connectivity pathways using chemical shift-dependent, apparent 1J(1H,13C): HNCACBcodedHAHB.
The proposed three-dimensional triple-resonance experiment HNCACBcodedHAHB correlates sequential 15N, 1H moieties via the chemical shifts of 13Calpha, 13Cbeta, 1Halpha, and 1Hbeta. The four sequential correlation pathways are achieved by the incorporation of the concept of chemical shift-coding [J. Biomol. NMR 25 (2003) 281] to the TROSY-HNCACB experiment. The monitored 1Halpha and 1Hbeta chemical shifts are then coded in the line shape of the cross-peaks of 13Calpha, 13Cbeta along the 13C dimension through an apparent residual scalar coupling, the size of which depends on the attached hydrogen chemical shift. The information of four sequential correlation pathways enables a rapid backbone assignment. The HNCACBcodedHAHB experiment was applied to approximately 85% labeled 13C,15N-labeled amino-terminal fragment of Vaccinia virus DNA topoisomerase I comprising residues 1-77. After one day of measurement on a Bruker Avance 700 MHz spectrometer and 8 h of manual analysis of the spectrum 93% of the backbone assignment was achieved.
Pegan
Scott
S
Structural Biology Laboratory, The Salk Institute, La Jolla, CA 92037, USA.
Kwiatkowski
Witek
W
Choe
Senyon
S
Riek
Roland
R
D000465:Algorithms; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry*; D002247:Carbon Isotopes; D003461:Crystallography / Q000379:methods*; D004264:DNA Topoisomerases, Type I / Q000032:analysis / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D013113:Spin Labels; D014616:Vaccinia virus / Q000737:chemistry / Q000201:enzymology
D000596:Amino Acids; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D013113:Spin Labels; D004264:DNA Topoisomerases, Type I