High-resolution 3D CANCA NMR experiments for complete mainchain assignments using C(alpha) direct detection.
The primary limitation of solution state NMR with larger, highly dynamic, or paramagnetic systems originates from signal losses due to fast transverse relaxation. This is related to the high gyromagnetic ratio gamma of protons, which are usually detected. Thus, it is attractive to consider detection of nuclei with lower gamma, such as (13)C, for extending the size limits of NMR. Here, we present an approach for complete assignment of C(alpha) and N resonances in fast relaxing proteins using a C(alpha) detected 3D CANCA experiment for perdeuterated proteins. The CANCA experiment correlates alpha carbons with the sequentially adjacent and succeeding nitrogen and alpha carbons. This enables elongation of the chain of assigned residues simply by navigating along both nitrogen and carbon dimensions using a "stairway" assignment procedure. The simultaneous use of both C(alpha) and N sequential connectivities makes the experiment more robust than conventional 3D experiments, which rely solely on a single (13)C indirect dimension for sequential information. The 3D CANCA experiment, which is very useful for mainchain assignments of higher molecular weight proteins at high magnetic field, also provides an attractive alterative for smaller proteins. Two versions of the experiment are described for samples that are (13)C labeled either uniformly or at alternate positions for removing one-bond (13)C-(13)C couplings. To achieve both high resolution and sensitivity, extensive nonuniform sampling was employed. Adding longitudinal relaxation enhancement agents can allow for shorter recycling delays, decreased measuring time, or enhanced sensitivity.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Koh
K
Takeuchi
Dominique P
DP
Frueh
Sven G
SG
Hyberts
Zhen-Yu J
ZY
Sun
Gerhard
G
Wagner
D002247:Carbon Isotopes; D009682:Magnetic Resonance Spectroscopy / Q000592:standards; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011789:Quantum Theory; D012015:Reference Standards
D002247:Carbon Isotopes; C029152:G-substrate; D009419:Nerve Tissue Proteins