Ansari, M.Z. and Kumar, A. and Ahari, D. and Priyadarshi, A. and Padmavathi, L. and Bhandari, Rashna and Swaminathan, R. (2018) Protein Charge Transfer Absorption Spectra: An Intrinsic Probe to Monitor Structural and Oligomeric Transitions in Proteins. Faraday Discussions, 207. pp. 91-113. ISSN 1359-6640

Text FD 2018.pdf Restricted to Repository staff only Download (1327Kb) | Request a copy

Abstract

Protein Charge Transfer Spectra (ProCharTS) originate when charged amino/carboxylate groups in the side chains of Lys/Glu, act as electronic charge acceptors/donors for photoinduced charge transfer either from/to the polypeptide backbone or to each other. The absorption band intensities in ProCharTS at wavelengths 250—800 nm are dependent on 3D spatial proximity between these charged functional groups across the protein. Intrinsically disordered proteins (IDPs) are an important class of proteins involved in signalling and regulatory functions in the eukaryotic cell. 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 (like PEST fragment of human c-Myc, its mutant and Dehydrin from maize) to sense structural transitions in IDPs using ProCharTS absorption spectra. Conformational changes induced in the protein by altering pH and 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. Results revealed that in presence of Trp/Tyr, ProCharTS absorbance was substantially reduced, specifically at wavelengths where absorption by Trp or Tyr was near its maximum. 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. Presence of 0.25 M NaCl or KCl in the medium also altered the ProCharTS spectrum. Finally, increase in ProCharTS absorbance with time, in HEWL in 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 as a label-free intrinsic probe to monitor changes in protein charge, structure and oligomeric state.

Item Type:	Article
Depositing User:	Users 2 not found.
Date Deposited:	28 Nov 2017 18:49
Last Modified:	17 Apr 2018 19:05
URI:	http://cdfd.sciencecentral.in/id/eprint/814

Actions (login required)

View Item