A thermodynamic model for interpreting tryptophan excitation-energy-dependent fluorescence spectra provides insight into protein conformational sampling and stability

Abstract

It is now over thirty 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. Whilst 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 protein 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 multi-tryptophan proteins, with examples including a structurally disordered peptide, de novo designed enzyme, human regulatory protein, therapeutic monoclonal antibody 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 bimolecular 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 $$$ \mathrm{CSM}\left({\lambda}_{\mathrm{Ex}}^{\mathrm{Fc}}\right) $$$ and ∆ G m that allow for biologically relevant insights to be drawn from the data (Figures 3b and S3) (Kwok et al 2021). Specifically, $$$ \mathrm{CSM}\left({\lambda}_{\mathrm{Ex}}^{\mathrm{Fc}}\right) $$$ provides information about the degree of tryptophan residue exposure to the surrounding solvent (a measure of protein foldedness) and ∆ G m describes the rigidity of the protein, which is linked to the aggregation state.…”

“…The value of the center of spectral mass (CSM) of the completely relaxed state of emission ($$$ \mathrm{CSM}\left({\lambda}_{\mathrm{Ex}}^R\right) $$$) was obtained by global fitting of data collected between $$$ {\lambda}_{\mathrm{Ex}} $$$ = 292–310 nm (Kwok et al 2021) and was 418.9 ± 3 nm for our trastuzumab variants. Individual variant fits all displayed regression p ‐values of less than 0.05 (Figure S3).…”

“…Only allelic sequences with non‐synonymous mutations were selected (Table S1). To meet the requirements of the REES technique (Knight et al 2020; Kwok et al 2021), we selected alleles within each subclass containing equal numbers of tryptophan residues. IGHG4 alleles were excluded from analysis due to incomplete sequence information in the CH1 domain or the presence of synonymous mutations.…”

“…where f i is the measured fluorescence intensity and λ Em is the emission wavelength (Knight, 2020). The CSM data was then fit with a thermodynamic model of REES behavior that describes a two‐state transition of tryptophan fluorophores from an excited to a relaxed state (Kwok et al 2021) (Equation ): $$$$ \mathrm{CSM}\left({\lambda}_{\mathrm{Ex}}\right)=\kern0.5em \frac{\mathrm{CSM}\left({\lambda}_{\mathrm{Ex}}^{\mathrm{Fc}}\right)+\mathrm{CSM}\left({\lambda}_{\mathrm{Ex}}^R\right){e}^{\left(\frac{\Delta Gm\left({\lambda}_{\mathrm{Ex}}-{\lambda}_{\mathrm{Ex}}^{50\%}\right)}{RT}\right)}}{1+{e}^{\left(\frac{\Delta Gm\left({\lambda}_{\mathrm{Ex}}-{\lambda}_{\mathrm{Ex}}^{50\%}\right)}{RT}\right)}} $$$$ …”

“…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).…”