The red edge excitation shift phenomenon can be used to unmask protein structural ensembles: implications for NEMO–ubiquitin interactions

Abstract

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-jB 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. Combination of 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 ligandbinding 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-ubiquitin 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.

A complete thermodynamic analysis of enzyme turnover links the free energy landscape to enzyme catalysis

“…a more flexible protein . The REES effect arises from photoselection of discrete conformational states reflected by differing solvent–solute interaction energies and has been described in depth recently . The effect manifests as a red shift in emission spectra (increased inhomogenous broadening) as the energy of the exciting photons is decreased (Fig.…”

“…That is, a larger REES effect reflects an increase in number of discrete species within the available equilibrium of conformational states, i.e. a more flexible protein . The REES effect arises from photoselection of discrete conformational states reflected by differing solvent–solute interaction energies and has been described in depth recently .…”

“…We fit the experimental REES data (Fig. B) to a phenomenological model based on an exponential function, similar to our previous fitting, primarily to capture the curvature that is always apparent in protein REES data sets .…”

“…a more flexible protein [24]. The REES effect arises from photoselection of discrete conformational states reflected by differing solvent-solute interaction energies and has been described in depth recently [24][25][26][27].…”

Self‐Assembly of PEGylated Diphenylalanines into Photoluminescent Fibrillary Aggregates

“…Both the systems are associated to a similar Stoke's shift (5602 cm À 1 for PEG2-FF and 5288 cm À 1 for PEG6-FF), calculated recording the excitation spectrum for each sample in the maximum of emission (spectra in Figure 6c). [41] The plot of emission maximum as function of the excitation wavelength is reported for both the FF derivatives in Figure 6d. On the evidence that the fluorescence quantum yield (φ) of phenylalanine residue is extremely low (φ = 0.020 at 303 K) [40] the detected PL emission can recommend an aggregation induced emission (AIE) phenomenon.…”