Dynamic Insight into Protein Structure Utilizing Red Edge Excitation Shift

Abstract

Proteins are considered the workhorses in the cellular machinery. They are often organized in a highly ordered conformation in the crowded cellular environment. These conformations display characteristic dynamics over a range of time scales. An emerging consensus is that protein function is critically dependent on its dynamics. The subtle interplay between structure and dynamics is a hallmark of protein organization and is essential for its function. Depending on the environmental context, proteins can adopt a range of conformations such as native, molten globule, unfolded (denatured), and misfolded states. Although protein crystallography is a well established technique, it is not always possible to characterize various protein conformations by X-ray crystallography due to transient nature of these states. Even in cases where structural characterization is possible, the information obtained lacks dynamic component, which is needed to understand protein function. In this overall scenario, approaches that reveal information on protein dynamics are much appreciated. Dynamics of confined water has interesting implications in protein folding. Interfacial hydration combines the motion of water molecules with the slow moving protein molecules. The red edge excitation shift (REES) approach becomes relevant in this context. REES is defined as the shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of absorption spectrum. REES arises due to slow rates (relative to fluorescence lifetime) of solvent relaxation (reorientation) around an excited state fluorophore in organized assemblies such as proteins. Consequently, REES depends on the environment-induced motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. In the case of a protein, the confined water in the protein creates a dipolar field that acts as the solvent for a fluorophore in the protein. In this Account, we focus on REES to monitor organization and dynamics of soluble and membrane proteins utilizing intrinsic protein fluorescence. We discuss here the application of REES in various conformations of proteins. While application of REES to proteins in native conformation has been in use for a long time, our work highlights the potential of this approach in case of molten globule and denatured conformations. For example, we have demonstrated the presence of residual structure, that could not be detected using other methods, by REES of denatured spectrin. Given the functional relevance of such residual structures, these results are very far reaching. We discuss here the application of REES to molten globule conformation and to the green fluorescent protein (GFP). The case of GFP is particularly interesting since the dipolar field in this case is provided by the protein matrix itself and not confined water. We envision that future applications of REES in proteins will involve generating a dynamic hydration map of the protein, which would allow us to explore protein function in terms of local dynamics and hydration.