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.

Background: NF-B essential modulator (NEMO) is the key regulator of NF-B signaling, but how it performs this regulation is poorly understood. Results: NEMO is a highly flexible protein that shows ligand-specific conformational change. Conclusion: Unanchored polyubiquitin binding controls NEMO conformational state and functional activity. Significance: Our findings provide a detailed mechanistic understanding of NEMO activity and develop the non-degradative role of free polyubiquitin.

The proteostasis network allows organisms to support and regulate the life cycle of proteins. Especially regarding stress, molecular chaperones represent the main players within this network. Small heat shock proteins (sHsps) are a diverse family of ATP-independent molecular chaperones acting as the first line of defense in many stress situations. Thereby, the promiscuous interaction of sHsps with substrate proteins results in complexes from which the substrates can be refolded by ATP-dependent chaperones. Particularly in vertebrates, sHsps are linked to a broad variety of diseases and are needed to maintain the refractive index of the eye lens. A striking key characteristic of sHsps is their existence in ensembles of oligomers with varying numbers of subunits. The respective dynamics of these molecules allow the exchange of subunits and the formation of hetero-oligomers. Additionally, these dynamics are closely linked to the chaperone activity of sHsps. In current models a shift in the equilibrium of the sHsp ensemble allows regulation of the chaperone activity, whereby smaller oligomers are commonly the more active species. Different triggers reversibly change the oligomer equilibrium and regulate the activity of sHsps. However, a finite availability of high-resolution structures of sHsps still limits a detailed mechanistic understanding of their dynamics and the correlating recognition of substrate proteins. Here we summarize recent advances in understanding the structural and functional relationships of human sHsps with a focus on the eye-lens αA- and αB-crystallins.

energy-dependent molecular beacon detects early stage neurotoxic A aggregates in the presence of cortical neurons. ACS β Chemical Neuroscience 10 (3) , pp.

Most biomolecular interactions are typically thought to increase the (local) rigidity of a complex, for example, in drug‐target binding. However, detailed analysis of specific biomolecular complexes can reveal a more subtle interplay between binding and rigidity. Here, we focussed on the human leucocyte antigen (HLA), which plays a crucial role in the adaptive immune system by presenting peptides for recognition by the αβ T‐cell receptor (TCR). The role that the peptide plays in tuning HLA flexibility during TCR recognition is potentially crucial in determining the functional outcome of an immune response, with obvious relevance to the growing list of immunotherapies that target the T‐cell compartment. We have applied high‐pressure/temperature perturbation experiments, combined with molecular dynamics simulations, to explore the drivers that affect molecular flexibility for a series of different peptide–HLA complexes. We find that different peptide sequences affect peptide–HLA flexibility in different ways, with the peptide cargo tuning a network of correlated motions throughout the pHLA complex, including in areas remote from the peptide‐binding interface, in a manner that could influence T‐cell antigen discrimination.