Integrative structure biology

 

The most important technique for structural biology in solution is Nuclear Magnetic Resonance Spectroscopy (NMR). However, the standard NMR methodology reaches its limit when applied to high-molecular-weight complexes, where the large line-width of NMR resonances impedes an accurate structure determination. It is common sense that NMR “does not work” for molecules >50 kDa.

As an NMR-expert laboratory, we believe that NMR has a yet unexploited potential for application to high-molecular-weight RNP assemblies. In the past decade clever methodological developments from the group of Lewis Kay in Canada broke the paradigm of NMR as a structural technique for “small proteins” (1). The methyl TROSY (Transverse Relaxation Optimized Spectroscopy) experiment, developed by this group, opened the way to studies of soluble proteins up to a molecular weight (MW) of 700-800 kDa and for concentrations as low as a few tens of mmolar. With the combination of methyl TROSY and the PRE (Paramagnetic Relaxation Enhancement) method (2) to measure inter-domain or intermolecular distances, the structure of large protein assemblies has come into reach by solution-state NMR.

Methyl TROSY cannot be applied to RNA, which does not contain any methyl groups. In addition, the number of structural restraints that can be obtained by the combination of methyl TROSY and PRE experiments is an order of magnitude smaller than that typically obtained from medium size proteins by standard NMR methodology. Both facts result in insufficient data for the determination of the structure of large RNP complexes by NMR.

Figure 1. Schematic representation of our integrative structural biology approach. The structure of RNP complexes in non-crystalline state results from hybrid data including NMR, SAS, EPR,  FRET,  EM and biochemical probing (Figure by Dr. Karaca).

 

To solve this problem, our laboratory seeks to compensate the limitations of individual methods by a combination of complementary structural techniques applicable to non-crystalline samples. Our philosophy is to tackle the structure of high-molecular-weight complexes, whose large size impedes a detailed structural description by NMR only, with an array of different complementary methodologies, such as segmental and specific labeling of both proteins and RNAs, small angle scattering (SAS), electron microscopy (EM), Electron Paramagnetic Resonance (EPR), Fluorescence Resonance Energy Transfer (FRET), mutational analysis and biochemical experiments (e.g. cross-link) (Fig. 1). The ensemble of structural data obtained by the different methods drives a molecular docking protocol that uses the high-resolution structures of sub-components of the complex to reconstitute the three-dimensional structure of the multi-component, high-molecular-weight object. With our complementary approach it is possible to examine RNP particles in solution, in their native environment, where they preserve both their structure and dynamic properties.

References

1.         Rosenzweig, R. and Kay, L.E. (2014) Bringing dynamic molecular machines into focus by methyl-TROSY NMR. D - 2985150r, 83, 291-315.

2.         Battiste, J.L. and Wagner, G. (2000) Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data. Biochemistry, 39, 5355-5365.