Automated projection spectroscopy and its applications.

ABSTRACT

This chapter presents the NMR technique APSY (automated projection spectroscopy) and its applications for sequence-specific resonance assignments of proteins. The result of an APSY experiment is a list of chemical shift correlations for an N-dimensional NMR spectrum (N≥3). This list is obtained in a fully automated way by the dedicated algorithm GAPRO (geometric analysis of projections) from a geometric analysis of experimentally recorded, low-dimensional projections. Because the positions of corresponding peaks in multiple projections are correlated, thermal noise and other uncorrelated artifacts are efficiently suppressed. We describe the theoretical background of the APSY method and discuss technical aspects that guide its optimal use. Further, applications of APSY-NMR spectroscopy for fully automated sequence-specific backbone and side chain assignments of proteins are described. We discuss the choice of suitable experiments for this purpose and show several examples. APSY is of particular interest for the assignment of soluble unfolded proteins, which is a time-consuming task by conventional means. With this class of proteins, APSY-NMR experiments with up to seven dimensions have been recorded. Sequence-specific assignments of protein side chains in turn are obtained from a 5D TOCSY-APSY-NMR experiment.