Dynamic structural science: exploring energy landscapes in complex enzyme systems

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Proteins are dynamic molecules. Motions within a protein molecule can be localised (e.g. bond vibrations, backbone and side chain motions) occuring on relatively fast timescales, or large scale motions (domain motions, conformational changes and slow breathing modes) that typically occur on the millisecond to second timescale. Localised fast motions can influence the chemistry in enzyme active sites; larger scale motions bring active sites together, or facilitate long range communication, for example in the transfer of electrons over large distances in redox enzymes, information transfer through signalling cascades or the folding of protein molecules. These large scale motions give rise to the concept of energy landscapes - that is the free energy surface that accommodates all conformations of the protein macromolecule that are populated. The distribution of conformational states across this landscape can be perturbed, for example by ligand or drug binding, natural variation in sequence (polymorphisms) or partner protein binding. Our knowledge of the spatial distribution and temporal exploration of these landscapes is at best limited, attributed in the main to the lack of general structural and biophysical tools to capture this information. The three dimensional structures of 'rigid' protein modules are readily accessed using conventional approaches (crystallography, NMR spectroscopy). How multiple modules communicate in complex protein systems however is not accessible using these techniques. In this application we aim to develop robust experimental methods using state-of-the-art spectroscopic, kinetic and computational methods that enable investigators to study the spatial and temporal properties of landscapes and their remodelling by small molecule/protein binding. We aim to develop these methods using mammalian nitric oxide synthases, redox enzymes that are constructed from multiple functional domain the chemistry of which is coupled to major dynamical excursions during the course of the enzyme catalysed reaction. We aim to define the structures of multiple conformational states across the landscape, define the timeconstants for their interconversion and assess the functional importance of these structural transitions in the catalytic cycle of the enzyme. By providing atomic level spatial and time resolved information on the functional dynamics in nitric oxide synthase enzymes we envisage that new opportunities will accrue to develop selective inhibitors that interfere with dynamical processes linked to function. This will reinvigorate the search for isoform specific inhibitors of these enzymes, and also provide general tools for similar analysis of other dynamic systems from which function and therapeutic intervention can be studied.