Protein Charge Transfer Absorption Spectra: An Intrinsic Probe to Monitor Structural and Oligomeric Transitions in Proteins

Abstract

Protein Charge Transfer Spectra (ProCharTS) originate when charged amino/carboxylate groups in Lys/Glu, act as electronic charge acceptors/donors for photoinduced electron transfer either from/to the polypeptide backbone or to each other (Prasad et. al., 2017, DOI: 10.1039/c7sc00880e). The absorption band intensities in ProCharTS (250—800 nm) are dependent on 3D spatial proximity between charged functional groups across the protein. Intrinsically disordered proteins (IDPs) are rich in charged amino acids, but lack structure promoting intrinsic spectral probes like Tyr or Trp in their sequence, making their structural characterisation difficult. Here, we exploit, the richness of charged amino acid population among IDPs to sense structural transitions among multiple IDPs (like PEST fragment of human c-Myc, its mutant and Dehydrin from maize) using ProCharTS absorption spectra. Conformational changes induced in the IDP by altering pH/temperature of aqueous medium was monitored by ProCharTS and confirmed by CD spectra. Further, utility of ProCharTS to detect protein aggregation was examined using Hen Egg-White Lysozyme (HEWL) protein. Significant changes in ProCharTS spectrum was observed with changing pH in the range 3—11, which correlated with changes in secondary structure of PEST fragment. Importantly, absorbance at 280 nm, which is often employed as a measure of protein concentration, was profoundly altered by changes in ProCharTS intensity in response to changing pH in Dehydrin. ProCharTS intensity was sensitive to temperature induced changes in the secondary structure of PEST fragments between 25—85oC. Finally, increase in ProCharTS absorbance with time, in HEWL at pH 2, directly correlated with growth of HEWL aggregates and amyloid fibrils as confirmed by rising thioflavin T fluorescence. Taken together, our work highlights the utility of ProCharTS absorption as a novel label-free intrinsic probe to monitor changes in protein charge, structure and oligomeric state.