Abstract
Conformational stability of a protein is usually obtained by spectroscopically measuring the unfolding melting temperature. However, optical spectra under native conditions are considered to contain too little resolution to probe protein stability. Here, we have built and trained a neural network model to take the temperature-dependence of intrinsic fluorescence emission under native-only conditions as inputs, and then predict the spectra at the unfolding transition and denatured state. Application to a therapeutic antibody fragment demonstrates that thermal transitions obtained from the predicted spectra correlate highly with those measured experimentally. Crucially, this work reveals that the temperature-dependence of native fluorescence spectra contains a high-degree of previously hidden information relating native ensemble features to stability. This could lead to rapid screening of therapeutic protein variants and formulations based on spectroscopic measurements under non-denaturing temperatures only.
Highlights
The measurement of conformational stability is crucial in protein folding study, as well as for the engineering and formulation development of protein-based therapeutics [1,2,3]
The denaturation of Fab results in the exposure of the tryptophan residues that lead to a change in fluorescence intensity as a function of temperature
The precise the midpoint (Tm) value obtained is affected by the experimental settings, the ramp rate of the thermal denaturation, which is kept constant across all experiments
Summary
The measurement of conformational stability is crucial in protein folding study, as well as for the engineering and formulation development of protein-based therapeutics [1,2,3]. The thermodynamic stability of proteins is often determined by monitoring the fraction of protein unfolding as a function of step-wise increases in temperature or chemical denaturant, giving rise to a transition phase that defines a thermal- or chemicaldenaturation mid-point (Tm or Cm) [4]. These measures are often used as rapid screens for improving the conformational stability of proteins through mutagenesis or formulation of the buffer conditions, aiming at a protein variant or formulation with improved kinetic stability to unfolding or aggregation at a given storage temperature. Mutations of an antibody fragment led to improved aggregation kinetics in cases that
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