To understand complex molecular interactions it is necessary to account for molecular flexibility and the available equilibrium of conformational states. Only a small number of experimental approaches can access such information. Potentially steady-state red edge excitation shift (REES) spectroscopy can act as a qualitative metric of changes to the protein free energy landscape (FEL) and the equilibrium of conformational states. First we validate this hypothesis using a single Trp containing protein, NF-κB essential modulator (NEMO). We provide detailed evidence from chemical denaturation studies, macromolecular crowding studies and the first report of the pressure-dependence of the REES effect. Combined these data demonstrate that the REES effect can report on the 'ruggedness' of the FEL and we present a phenomenological model, based on realistic physical interpretations, for fitting steady-state REES data to allow quantification of this aspect of the REES effect. We test the conceptual framework we have developed by correlating findings from NEMO ligand binding studies with the REES data in a range of NEMO-ligand binary complexes. Our findings shed light on the nature of the interaction between NEMO and poly-ubiquitin, suggesting that NEMO is differentially regulated by poly-ubiqutin chain length and that this regulation occurs via a modulation of the available equilibrium of conformational states, rather than gross structural change. This study therefore demonstrates the potential of REES as a powerful tool for tackling contemporary issues in structural biology and biophysics and elucidates novel information on the structure-function relationship of NEMO and key interaction partners. This article is protected by copyright. All rights reserved.
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- Department of Biology & Biochemistry - Senior Lecturer
- Centre for Sustainable and Circular Technologies (CSCT)
- Centre for Biosensors, Bioelectronics and Biodevices (C3Bio)
- Centre for Therapeutic Innovation
- Centre for Integrated Bioprocessing Research (CIBR)
Person: Research & Teaching