Carbonyl carbon label selective (CCLS) 1H-15N HSQC experiment for improved detection of backbone 13C-15N cross peaks in larger proteins.
We present a highly sensitive pulse sequence, carbonyl carbon label selective (1)H-(15)N HSQC (CCLS-HSQC) for the detection of signals from (1)H-(15)N units involved in (13)C'-(15)N linkages. The CCLS-HSQC pulse sequence utilizes a modified (15)N CT evolution period equal to 1/( [Formula: see text]) ( approximately 33 ms) to select for (13)C'-(15)N pairs. By collecting CCLS-HSQC and HNCO data for two proteins (8 kDa ubiquitin and 20 kDa HscB) at various temperatures (5-40 degrees C) in order to vary correlation times, we demonstrate the superiority of the CCLS-HSQC pulse sequence for proteins with long correlation times (i.e. higher molecular weight). We then show that the CCLS-HSQC experiment yields assignments in the case of a 41 kDa protein incorporating pairs of (15)N- and (13)C'-labeled amino acids, where a TROSY 2D-HN(CO) had failed. Although the approach requires that the (1)H-(15)N HSQC cross peaks be observable, it does not require deuteration of the protein. The method is suitable for larger proteins and is less affected by conformational exchange than HNCO experiments, which require a longer period of transverse (15)N magnetization. The method also is tolerant to the partial loss of signal from isotopic dilution (scrambling). This approach will be applicable to families of proteins that have been resistant to NMR structural and dynamic analysis, such as large enzymes, and partially folded or unfolded proteins.
Tonelli
Marco
M
Department of Biochemistry, National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.
Masterson
Larry R
LR
Hallenga
Klaas
K
Veglia
Gianluigi
G
Markley
John L
JL
D002247:Carbon Isotopes; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results