Homonuclear Super-Resolution NMR Spectroscopy

Abstract

In homonuclear ¹H NMR (nuclear magnetic resonance) spectra such as [¹H,¹H]-NOESY (Nuclear Overhauser Enhancement spectroscopy), which is a historic cornerstone spectrum for biomolecular NMR structural biology, hundreds to thousands of cross peaks are present within a square of approximately 100 ppm² leading to a lot of signal overlap. Spectral resolution is thus a limiting factor for unambiguous chemical shift assignment and data interpretation for dynamics and structure elucidation. Acquiring the spectra at higher magnetic fields such as at a 1.2 GHz ¹H frequency helps to reduce spectral crowding, since resolution scales proportionally to the magnetic field strength. Here, we show that the linewidths of cross peaks in [¹H,¹H]-NOESY and [¹H,¹H]-TOCSY spectra can be further reduced by a factor of 2–3 in each dimension by super-resolution spectroscopy. In the indirect dimension a composite exponential-cosine weighted number of scans along the time increments are recorded and digitally smoothened by a window function, while in the direct dimension an exponential-cosine window function is applied. Furthermore, measurement time saving by reduced-acquisition super-resolution (RASR) is introduced. Application to the 20 kDa protein KRAS shows that highly resolved NMR spectra suitable for automated analysis can be acquired within less than 3 hours. The method opens an avenue towards automated chemical shift assignment, dynamics and structure determination of unlabeled small and medium size proteins within 24 hours.