Side-chain H and C resonance assignment in protonated/partially deuterated proteins using an improved 3D(13)C-detected HCC-TOCSY.
We propose the use of (13)C-detected 3D HCC-TOCSY experiments for assignment of (1)H and (13)C resonances in protonated and partially deuterated proteins. The experiments extend 2D C-13-start and C-13-observe TOCSY type experiments proposed earlier. Introduction of the third (1)H dimension to 2D TOCSY: (i) reduces the peak overlap and (ii) increases the sensitivity per unit time, even for highly deuterated (>85%) protein samples, which makes this improved method an attractive tool for the side-chain H and C assignment of average sized proteins with natural isotope abundance as well as large partially deuterated proteins. The experiments are demonstrated with a 16 kDa (15)N, (13)C-labeled non-deuterated apo-CcmE and a 48 kDa uniformly (15)N, (13)C-labeled and fractionally ( approximately 90%) deuterated dimeric sFkpA. It is predicted that this method should be suitable for the assignment of methyl (13)C and (1)H chemical shifts of methyl protonated, highly deuterated and (13)C-labeled proteins with even higher molecular weight.
Hu
Kaifeng
K
Laboratorium für Physikalische Chemie, Swiss Federal Institute of Technology, ETH-Hönggerberg, CH-8093 Zürich, Switzerland. kaifeng.hu@bionmr.phys.chem.ethz.ch
Vögeli
Beat
B
Pervushin
Konstantin
K
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002247:Carbon Isotopes; D003903:Deuterium; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry*; D020104:Immunophilins / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D019696:Peptidylprolyl Isomerase / Q000737:chemistry*; D011487:Protein Conformation; D012815:Signal Processing, Computer-Assisted
D001425:Bacterial Outer Membrane Proteins; D002247:Carbon Isotopes; C115384:CcmE protein, E coli; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D008565:Membrane Proteins; D003903:Deuterium; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase