Recent Advances in Solution NMR Studies: 13C Direct Detection for Biomolecular NMR applications

Carbon-13 direct detected NMR experiments are widely used for the study of small molecules thanks to the great amount of information that can be obtained from the observed chemical shifts and coupling topologies. However, after the pioneering studies by the group of John Markley, 13C direct detection for biomolecular NMR applications in solution has been abandoned. A wide variety of experiments based on 1H detection, which exploit heteronuclei in the indirect dimensions, have been developed on the ground of the much higher intrinsic sensitivity of 1H with respect to that of 13C, and are routinely used. Therefore, why worry about 13C direct detection for biomolecular applications in solution? The reason is probably twofold. On one hand, the great improvement in instrumental sensitivity we have assisted to in the last decade, initially exploited to study systems with limited solubility, can also be exploited to investigate nuclear spins characterized by a smaller gyromagnetic ratio and thus lower sensitivity. On the other hand, the great progress in the hardware, the vast array of experimental methods that have been developed, as well as progress in sample preparation and isotopic labeling strategies, have opened the way to the characterization through NMR spectroscopy of systems of increasing complexity. Several areas in which 1H detected experiments find limitations (despite the higher sensitivity) have thus emerged, stimulating the design of alternative methods able to exploit the intrinsically different properties of the various nuclear spins. The fast progress in the implementation of hyperpolarization techniques might completely change our way of thinking in the next future with the intrinsic sensitivity of a specific nucleus playing a minor role in the choice of the specific approach! Most of the high-field high resolution NMR instruments are nowadays equipped with probeheads characterized by an excellent sensitivity not only for 1H, but also for 13C. A wide variety of multidimensional NMR experiments based on 13C direct detection have been developed for biomolecular NMR applications. It is thus timely to evaluate the progress and the perspectives in this field. In this review we will focus on the peculiar properties of the heteronuclei, and of 13C in particular, to identify the key features of heteronuclei that may result useful and complementary to the ones of protons (Figure 1). We will then summarize the many approaches proposed to overcome the problem of homonuclear couplings in the direct acquisition dimension that could potentially complicate the spectra and reduce the sensitivity. A survey of experiments that have been developed for a variety of different cases will then be presented, highlighting the many variants proposed. Finally, as biomolecular 13C direct detection in many cases is not limited any longer by sensitivity, we will present how all the approaches to reduce experimental time that have recently been implemented find application in the various 13C detected experiments. The suite of experiments developed can thus be used as a general complementary tool for biomolecular applications and it can provide additional unique information for different kinds of proteins, such as paramagnetic proteins, large multimeric assemblies as well as intrinsically disordered proteins.