HMQC and HSQC experiments with water flip-back optimized for large proteins.
An HMQC experiment is proposed, dubbed FHMQC, where water flip-back is achieved by a single water-selective pulse preceding the basic HMQC pulse sequence. The scheme is demonstrated with a 15N,1H-HMQC spectrum of uniformly 15N/2H-labelled S. aureus DNA gyrase B with a molecular weight of 45 kDa for the unlabelled protein. The sensitivity of the experiment is improved compared to that of an FHSQC spectrum. It is further shown that the original FHSQC experiment can be shortened by the use of bipolar gradients. Relaxation times of different 15N magnetizations and coherences were measured. The new FHMQC scheme is implemented in 3D NOESY-15N-HMQC and 3D 15N-HMQC-NOESY-15N-HMQC pulse sequences which are demonstrated with a 24 kDa fragment of uniformly 15N/13C/2H-labelled S. aureus DNA gyrase B.
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
P
P
Andersson
B
B
Gsell
B
B
Wipf
H
H
Senn
G
G
Otting
D001426:Bacterial Proteins / Q000737:chemistry*; D002247:Carbon Isotopes; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000737:chemistry; D003903:Deuterium; D007553:Isotope Labeling / Q000379:methods; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D014867:Water / Q000737:chemistry*
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D014867:Water; D003903:Deuterium; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II