A Thermodynamic Model for Interpreting Tryptophan Excitation-Energy-Dependent Fluorescence Spectra Provides Insight Into Protein Conformational Sampling and Stability

Abstract

It is now over 30 years since Demchenko and Ladokhin first posited the potential of the tryptophan red edge excitation shift (REES) effect to capture information on protein molecular dynamics. While there have been many key efforts in the intervening years, a biophysical thermodynamic model to quantify the relationship between the REES effect and protein flexibility has been lacking. Without such a model the full potential of the REES effect cannot be realized. Here, we present a thermodynamic model of the tryptophan REES effect that captures information on protein conformational flexibility, even with proteins containing multiple tryptophan residues. Our study incorporates exemplars at every scale, from tryptophan in solution, single tryptophan peptides, to multitryptophan proteins, with examples including a structurally disordered peptide, de novo designed enzyme, human regulatory protein, therapeutic monoclonal antibodies in active commercial development, and a mesophilic and hyperthermophilic enzyme. Combined, our model and data suggest a route forward for the experimental measurement of the protein REES effect and point to the potential for integrating biomolecular simulation with experimental data to yield novel insights.

Constant domain polymorphisms influence monoclonal antibody stability and dynamics

“…Fluorescent spectra were obtained before and after heating in a pH 8 buffer designed to induce destabilization over a timescale amenable to analysis in a single day (Cheng et al 2012) (Figure 3a). A recently developed thermodynamic model for REES was then fitted to the fluorescent data to extract values for CSM λ Fc Ex À Á and ΔG m that allow for biologically relevant insights to be drawn from the data (Figures 3b and S3) (Kwok et al 2021). Specifically, CSM λ Fc Ex À Á provides information about the degree of tryptophan residue exposure to the surrounding solvent (a measure of protein foldedness) and ΔG m describes the rigidity of the protein, which is linked to the aggregation state.…”

“…This highly sensitive approach detects distinct flexibility profiles for structurally identical proteins harboring single point mutation differences (Jones et al 2017). The underlying principles of REES have been thoroughly discussed in several publications (Catici et al 2016;Haldar et al 2011;Kwok et al 2021). In brief, REES exploits the fluorescent properties of tryptophan (Trp) residues as highly sensitive reporters of the local protein environment (Figure 1).…”

N-linked glycosylation increases horse radish peroxidase rigidity leading to enhanced activity and stability

“…As in our previous work [32][33][34][35] , we therefore numerically modelled each of the spectra using a sum of two skewed Gaussians (Eq 1) as described recently for a de novo haem peroxidase 33 𝑓𝑖 = 𝑓 ! "# exp (− ln 2) .…”

“…Fluorescence spectra were accurately modelled and deconvolved by fitting to such functions as demonstrated by elsewhere. [32][33][34][35] By fitting to a sum of two skewed Gaussians we were able to accurately model the spectral component attributable to tryptophan emission alone. From these models the centre of spectral mass (CSM) was extracted for each spectral component, which allowed quantification of changes in the structure of the fluorescence spectra,…”

“…As shown in Figure 1a, all 8 surface-exposed N-linked glycosylation sites are distant from the largely buried active site; the closest N-linked site to the haem centre is N214 at ~20Å. We have recently developed the use of an optical phenomenon that allows changes in the flexibility of a protein to be assessed via tryptophan fluorescence measurements: the red edge excitation shift (REES) 11,32,34,44 . REES is an optical phenomenon which tracks changes in the distribution of protein conformational states, via shifts in solvent-fluorophore [Trp] interaction energies 45 .…”