Methodological advancements for the measurement of residual anisotropic spin interactions in proteins by NMR spectroscopy

Residual anisotropic spin interactions, such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs), have the ability to provide valuable information about internuclear distances and orientations as well as molecular dynamics in proteins by NMR spectroscopy. The measurement of RDCs and RCSAs, however, presents many challenges, especially in higher molecular weight systems where their utility is further increased due to the use of extensive perdeuteration at non-labile proton sites to mitigate deleterious 1H-1H interactions, which limits the number of measureable proton-proton Nuclear Overhauser Effect (NOE) contacts. Still, even in the presence of perdeuteration, unfavorable relaxation in large proteins severely attenuates sensitivity and causes detrimental line broadening, which limits precision of measurement. Measurement of smaller (i.e. more distant) interactions is limited by the inability to employ sufficiently strong molecular alignment while still maintaining reasonable linewidths as well as the need to deconvolute their influence from that of the much larger close-in interactions. Therefore, it is desirable to develop new and improved methodologies for the measurement and analysis of RCAs and RDCs under adverse conditions with improved precision and accuracy.;To that end, new and innovative methods have been developed for the measurement of one-bond 1HN-15N RDCs and long range 1HN-13C' RDCs with improved precision. These include applications to medium protonated proteins (15-25 kDa) using frequency based 3D- HNCO methods and to large perdeuterated proteins (>25kDa) using frequency based 3D-TROSY-HNCO methods. To improve measurement smaller long range RDCs, a new double resonance technique for the inversion of single spin-coupled pairs inspired from Selective Single-Transition Cross-Polarization (ST-CP) Spectroscopy has been developed. This approach, combines successive phase-alternated and frequency-shifted selective Hartmann-Hahn matched cross-polarization contact pulses applied to 1H N and 15N to deliver highly efficient and selective inversion of the target 1HN resonance. This allows for the selective activation of RDC interactions within a specific spin-coupled network under conditions of strong alignment. Additionally, investigations into the contributions to observed anisotropic chemical shifts from alignment media solvent effects have made progress towards the improvement of the measurement of RCSAs.