Abstract
The access to weak alignment media has fuelled the development of methods for efficiently and accurately measuring residual dipolar couplings (RDCs) in NMR-spectroscopy. Among the wealth of approaches for determining one-bond scalar and RDC constants only J-modulated and J-evolved techniques retain maximum resolution in the presence of differential relaxation. In this article, a number of J-evolved experiments are examined with respect to the achievable minimum linewidth in the J-dimension, using the peptide PA4 and the 80-amino-acid-protein Saposin C as model systems. With the JE-N-BIRDd,X-HSQC experiment, the average full-width at half height could be reduced to approximately 5 Hz for the protein, which allows the additional resolution of otherwise unresolved peaks by the active (J+D)-coupling. Since RDCs generally can be scaled by the choice of alignment medium and alignment strength, the technique introduced here provides an effective resort in cases when chemical shift differences alone are insufficient for discriminating signals. In favorable cases even secondary structure elements can be distinguished.
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Acknowledgment
B.L. and H.K. thank the Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft (Emmy Noether fellowship LU 835/1-1; Ke 147/37-1) for financial support. We thank Martin Sukopp (Stanford University, USA) for the synthesis of PA4, Michaela Wendeler (National Cancer Institute, Frederick MD, USA) and Konrad Sandhoff (Universität Bonn, Germany) for kindly providing Saposin C and Jochen Klages (TU München, Germany) for help with the assignment of Saposin C.
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Appendix
Appendix
Neglecting strong coupling artifacts, the evolution of transverse operators during a BIRDd,X element applied to a three spin system consisting of one heteronucleus X, one directly coupled proton spin I d, and one remotely coupled proton spin I r with the corresponding couplings J d,X > J d,r ≈ J r,X , results in the following transfers:
If we assume that long-range heteronuclear and homonuclear couplings can be neglected during the BIRDd,X element, the transfers can be simplified to
for proton transverse operators and
for the corresponding transverse operators of the heteronucleus. The magnetization of every other operator is scaled by \({\cos(2\pi\delta^{1}J_{d,X})},\) resulting in an average loss of \({0.5^\ast(1- \cos(2\pi\delta^{1}J_{d,X}))}\) due to incomplete transfer during the BIRDd,X filter.
To examine the influence of homonuclear and long-range heteronuclear couplings, we assume a perfectly matched delay δ, leading to \({\hbox{cos}(2\pi\delta^{1}J_{d,X})=-1}\) and \({\sin(2\pi\delta^{1}J_{d,X})=0}.\) The transfers are then
and
As with the direct one-bond coupling, every other transverse component is scaled by the homonuclear coupling \({\cos(2\pi \delta ^{1}J_{d,r})},\) causing an average loss of magnetization of 0.5*(1- \({\cos(2\pi \delta ^{1}J_{d,r}))}.\) Heteronuclear long-range couplings, instead, do not affect proton transverse operators, but reduce all transverse operators on the heteronucleus according to \({(1-\cos(2\pi \delta ^{1}J_{r,X}))}.\)
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Furrer, J., John, M., Kessler, H. et al. J-Spectroscopy in the presence of residual dipolar couplings: determination of one-bond coupling constants and scalable resolution. J Biomol NMR 37, 231–243 (2007). https://doi.org/10.1007/s10858-006-9130-x
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DOI: https://doi.org/10.1007/s10858-006-9130-x