Measurement of one-bond 13Calpha-1Halpha residual dipolar coupling constants in proteins by selective manipulation of CalphaHalpha spins.
We have developed new 2D and 3D experiments for the measurement of C(alpha)-H(alpha) residual dipolar coupling constants in (13)C and (15)N labelled proteins. Two experiments, 2D (HNCO)-(J-CA)NH and 3D (HN)CO-(J-CA)NH, sample the C(alpha)-H(alpha) splitting by means of C(alpha) magnetization, while 2D (J-HACACO)NH and 3D J-HA(CACO)NH use H(alpha) magnetization to achieve a similar result. In the 2D experiments the coupling evolution is superimposed on the evolution of the (15)N chemical shifts and the IPAP principle is used to obtain (1)H-(15)N HSQC-like spectra from which the splitting is determined. The use of a third dimension in 3D experiments reduces spectral overlap to the point where use of an IPAP scheme may not be necessary. The length of the sampling interval in the J-dimension of these experiments is dictated solely by the relaxation properties of C(alpha) or H(alpha) nuclei. This was made possible by the use of C(alpha) selective pulses in combination with either a DPFGSE or modified BIRD pulses. Inclusion of these pulse sequence elements in the J-evolution periods removes unwanted spin-spin interactions. This allows prolonged sampling periods ( approximately 25 ms) yielding higher precision C(alpha)-H(alpha) splitting determination than is achievable with existing frequency based methods.
School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK.
Graeme
G
Ball
Nicola
N
Meenan
Krystyna
K
Bromek
Brian O
BO
Smith
Juraj
J
Bella
Dusan
D
Uhrín
D000465:Algorithms; D000485:Allergens / Q000737:chemistry*; D052179:Antigens, Plant; D002247:Carbon Isotopes; D015801:Helminth Proteins / Q000737:chemistry*; D006859:Hydrogen; D006860:Hydrogen Bonding; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted
C093689:ABA-1 protein, Ascaris suum; D000485:Allergens; D052179:Antigens, Plant; D002247:Carbon Isotopes; D015801:Helminth Proteins; D006859:Hydrogen