Our work spanned three workpackages concerning sample preparation, development of methods to study structure, dynamics and interactions of proteins in complexes and application of the developed approach to understand the factors responsible for controlling biosynthesis of useful natural products. 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 measurements at the same time ((J. Magn. Reson. 2019, 305, 219.). We have developed and validated 1H and 15N solvent Paramagnetic Relaxation Enhancements as a tool to probe protein-protein complexes in the solid state (J. Am. Chem. Soc. 2017, 139 (35), 12165.). 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, 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; (Sci. Rep. 2019, 9 (1), 1.)). 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 (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. In collaboration with Prof. V. Ladizhansky’s group (University of Guelph, Canada) we have also explored quantification of anisotropic protein motions in a membrane protein, sensory rhodopsin (J. Am. Chem. Soc. 2017, 139 (27), 9246.).
We have applied the developed approach in the context of integrated structural biology of protein complexes. In particular, we have studied the molecular level basis for specificity and directionality of antibiotic biosynthesis and modes of action for antibiotics. For example, we have elucidated the mechanism for chain termination in biosynthesis of enacyloxin, antibiotic active against multidrug resistant WHO priority pathogen Acinetobacter baumannii), and shown the suitability of the crucial to the process interaction between intrinsically disordered Short Linear Motif and beta-hairpin docking domain for engineering and creating hybrid systems to make new compounds (Nat. Chem. 2019, 11 (10), 913.). We have developed mechanistic understanding of the interactions between epimerisation domains and condensation domains in biosynthesis of antibiotic tyrocidine and further evaluated the suitability of the involved communication domains for protein engineering. A notable example of investigation for modes of action of antibiotic is our study on interactions of antibiotic teixobactin with the building block of bacterial wall, lipid II (Chem. Sci. 2018, 9 (47), 8850.), which has revealed that a composite fibril formation is behind the incredible efficiency of the antibiotic in question.
