Efficient acquisition of high-resolution 4-D diagonal-suppressed methyl–methyl NOESY for large proteins

Abstract

The methyl–methyl NOESY experiment plays an important role in determining the global folds of large proteins. Despite the high sensitivity of this experiment, the analysis of methyl–methyl NOEs is frequently hindered by the limited chemical shift dispersion of methyl groups, particularly methyl protons. This makes it difficult to unambiguously assign all of the methyl–methyl NOE crosspeaks using 3-D spectroscopy. The recent development of sparse sampling methods enables highly efficient acquisition of high-resolution 4-D spectra, which provides an excellent solution to resolving the degeneracy of methyl signals. However, many reconstruction algorithms for processing sparsely-sampled NMR data do not provide adequate suppression of aliasing artifacts in the presence of strong NOE diagonal signals. In order to overcome this limitation, we present a 4-D diagonal-suppressed methyl–methyl NOESY experiment specifically optimized for ultrasparse sampling and evaluate it using a deuterated, ILV methyl-protonated sample of the 42kDa Escherichia coli maltose binding protein (MBP). Suppression of diagonal signals removes the dynamic range barrier of the methyl–methyl NOESY experiment such that residual aliasing artifacts in the CLEAN-reconstructed high-resolution 4-D spectrum can be further reduced. At an ultrasparse sampling rate of less than 1%, we were able to identify and unambiguously assign the vast majority of expected NOE crosspeaks between methyl groups separated by less than 5Å and to detect very weak NOE crosspeaks from methyl groups that are over 7Å apart.