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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - complexNMR (Structural Dynamics of Protein Complexes by Solid-State NMR)

Teaser

Summary

Proteins in complexes perform most processes in the cell. In fact, most essential genes, i.e. genes critical for cell survival, encode for protein complexes. Frequently, interactions in assemblies lead to modification of structure and dynamics of proteins compared to their isolated states. Knowledge of such changes is often the key to understanding the mechanisms of protein function and malfunction, and can be achieved only through the study of proteins in molecular complexes rather than their isolated form. Only with such knowledge can we harness the power of natural processes for applications beneficial to human beings, e.g. by enabling synthetic biology approaches to produce new drugs to combat diseases or facilitate food production, and to correct malfunction of natural processes, e.g. design of inhibitors to affect a signalling pathway that is associated with a specific disease. However, large protein complexes with multiple components present very serious challenges to existing structural biology methods. There is a dire need of analytical methods that can overcome the existing challenges; complexNMR intends to fulfil this need.
The overarching goal of complexNMR is to develop a multidisciplinary approach involving solid and solution state NMR complemented by modelling to enable direct atomic resolution determination of the structure, dynamics and interactions of protein domains in large biomolecular complexes. Our approach paves the way towards a more holistic understanding of many processes occurring in the cell by simplifying studies of proteins, not only as isolated entities but as components of interacting complex systems.
In order to maximize applicability of our methods we aim them to be feasible with very small amounts of proteins.

Work performed

During the initial period of the project our work spanned all three workpackages with the main focus on achieving objectives from worpackages one and two, i.e. methodological part of the proposal. In particular, we concentrated on developing methods to study both structure and dynamics of large protein complexes paying special attention to approaches that enable working on very small samples (on the order of 2-20 nanomoles of the observed component). Large fraction of the efforts concerned sample preparation strategies, which are crucial to successful outcome of the project. We have extensively explored different techniques to speed up acquisition and get more information from smaller samples. Based on time-sharing approach and multiple receivers, we have developed a suite of experiments that allow recording several different spectra in a single experiment that facilitate spectral assignment and can be used to obtain multiple structural restraints at the same time (manuscript in preparation). We have developed and validated 1H and 15N solvent Paramagnetic Relaxation Enhancements as a tool to probe protein-protein complexes in the solid state (manuscript in preparation). We have used paramagnetic effects to develop a series of new fast experiments for probing protein dynamics: fast methods for measurement of dipolar order parameters at fast spinning frequencies (manuscript in preparation), accelerated relaxation dispersion approach that enabled quantitative characterisation of microsecond motions in large protein complexes (we demonstrated on the example of GB1:IgG complex that such measurements can be performed in 5 days instead of > 2 months as required with the existing approaches; manuscript in preparation). We have found out that relaxation measurements for protein GB1 in complex with IgG are consistent with a presence of an overall small amplitude anisotropic motion, where GB1 is sampling the binding interface (publication: Lamley, J. M.; Ã–ster, C.; Stevens, R. A. R. A.; Lewandowski, J. R. Angew. Chemie Int. Ed. 2015, 54 (51), 15374.). We have developed an approach for quantifying anisotropic peptide plane fluctuations based on analysis of combination of relaxation rates and dipolar order parameters (manuscript in preparation). In collaboration with Prof. V. Ladizhanskyâ€™s group (University of Guelph, Canada) we have also explored quantification of anisotropic protein motions in a membrane protein, sensory rhodopsin (manuscript in preparation).
Finally, we have made a good progress in application of multidisciplinary approaches to characterise complexes from polyketide synthases (PKSs) in order to determine molecular level basis for specificity and directionality of antibiotic biosynthesis. We have characterised both systems from enacyloxin PKS (manuscript in preparation) and gladiolin PKS (manuscript in preparation).

Final results

All the methods developed in the course are aimed at enabling new applications on complex and previously inaccessible systems. Importantly, the techniques are optimised for use on minute amounts of samples, which significantly extends their applicability. These methods significantly extend the state of the art for atomic resolution characterization of structure and dynamics of biological systems. Understanding of such systems at molecular level may lead to developing new solutions to combat a range of important societal problems, in particular in the context of health related challenges. To date the largest impact was achieved in the context of developing new antibiotics to combat multidrug resistant infections. For example, we have discovered a series of communication rules for the enzymes in polyketide synthases. These rules can be exploited to engineer viable hybrid systems that can be used to make new variants of antibiotics more suitable to be actual drugs in a so called synthetic biology approach.