BPS2026 – Protein CT spectra as a tool to reveal protein-protein, protein-nucleic acid, and post-translational events

Protein charge transfer spectra (ProCharTS) refers to UV-visible absorption in the 250–800 nm region among proteins that teem with charged amino acids, having clusters of ionized headgroups that lie within a vicinity of 3–8 Å of each other owing to the protein 3D fold. TD-DFT calculations on structure snapshots obtained from MD simulations have revealed that ProCharTS arises by photoinduced electron transfer from donors like polypeptide backbone (PPB); carboxylate anions of Asp/Glu to acceptors like cationic groups of Lys/Arg/protonated His; and PPB (10.1039/c7sc00880e). The molar extinction coefficient of the ProCharTS absorption spectrum is dependent on the charge and spatial proximity among the charged amino acid headgroups in the protein (10.1021/acs.jpcb.9b10071). Consequently, owing to such sensitivity, ProCharTS is a useful label-free tool to detect protein-DNA complex formation as we demonstrate for single chain IHF protein; protein-protein oligomer formation as we show for human lysozyme and α-synuclein; and protein post-translational modifications like acetylation and succinylation of α 3 C, α 3 W, H2A, and HSA proteins; dephosphorylation of α/β-casein proteins; and glycation of HSA. Interestingly, titration of protein against DNA and vice-versa reveals a distinct bell-shaped pattern of fractional increase in ProCharTS intensity for consensus DNA, while a flat profile is observed for non-consensus DNA. Both α and β-casein displayed an increase in molar extinction coefficient in the 350–600 nm region with increasing dephosphorylation. The dephosphorylation of five phosphates in β-casein and eight phosphates in α-casein were clearly detectable using ProCharTS. Increase in ProCharTS absorption displayed a biphasic profile with time upon aggregation of α-synuclein. Further, ProCharTS was sensitive enough to detect addition of one succinyl group to the α 3 C protein in contrast to acetylation, where addition of five acetyl groups was necessary to see noticeable change.