Most proteins have defined structures to bind with other protein molecules in cells or perform other functions such as catalysis. Intrinsic tryptophan (Trp) fluorescence spectroscopy has been an established technique to monitor protein conformational changes and protein-ligand interactions in solution but limited to rely on the information of emission intensity, anisotropy, time-resolved or fluorescence resonance energy transfer (FRET). The resolution of protein-protein and protein-solvent interactions in real-time may have been overlooked. The Red Edge Excitation Shift (REES) has been observed and described as a fluorescence phenomenon that a fluorophore excited by the photon near to the red edge of the spectrum displays its emission maximum shifting to the longer wavelength because of solvent relaxation. Such REES phenomena, which are characterised by inhomogeneous broadening and site photoselection, have been seldom used for studying protein dynamics. To further understand the red edge effect in protein dynamics, protein stability, folding and unfolding mechanism, we use REES incorporated with a new spectroscopic analytical model, Quantitative Understanding of Bio-molecular Edge Shift (QUBES) as a tool to explore and investigate the dynamics of Trp in solution, protein-ligand binding of alpha synuclein (α-syn) mutants labelled with single Trp, protein flexibility of a hyper-thermophilic enzyme and aggregation of multiple-Trp antibodies. In the present study, we verified and defined the meanings of the empirical values from the REES model. We demonstrated the CSM0 shifted to blue and Trp in phosphate-methanol buffer showed solvatochromic effect when the dielectric constant decreased. The specific site photoselection of REE effect was used to predict the metal binding site on α-syn mutants labelled with single Trp. This was further approved by syn-metal-therapeutic peptide binding, the therapeutic peptide was designed to stop the aggregates formation of syn-metal binding. The REE and the QUBES also showed the difference of the rigidity of multiple Trp enzymes, maltose-inducible α-glucosidase (MalL) and its mutant (V200S) temperature dependence without conformational change. In addition, REE and QUBES models provided extremely sensitive information of the presence of aggregates in multi-Trp monoclonal antibodies temperature dependence study. The high percentage of detecting impurities or aggregates in therapeutic monoclonal antibodies aids the pharmaceutical industries to predict and improve the development, the shelf-life and the efficacy of their biologics.
Evaluating the information content of the protein red edge excitation shift phenomenon: (Alternative Format Thesis)
Kwok, A. (Author). 23 Jul 2025
Student thesis: Doctoral Thesis › PhD