Identification of protein secondary structures by laser induced autofluorescence: A study of urea and GnHCl induced protein denaturation

Siddaramaiah, Manjunath and Satyamoorthy, K and Rao, Satish BS and Roy, Suparna and Chandra, Subhash and Mahato, Krishna Kishore (2017) Identification of protein secondary structures by laser induced autofluorescence: A study of urea and GnHCl induced protein denaturation. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 174 (1). pp. 44-53. ISSN 1386-1425

[img] PDF
RMS - 2612.pdf - Published Version
Restricted to Registered users only

Download (155kB) | Request a copy

Abstract

In the present study an attempt has beenmade to interrogate the bulk secondary structures of someselected proteins (BSA, HSA, lysozyme, trypsin and ribonuclease A) under urea and GnHCl denaturation using laser induced autofluorescence. The proteins were treated with different concentrations of urea (3 M, 6 M, 9 M) and GnHCl (2 M, 4 M, 6 M) and the corresponding steady state autofluorescence spectra were recorded at 281 nm pulsed laser excitations. The recorded fluorescence spectra of proteins were then interpreted based on the existing PDB structures of the proteins and the Trp solvent accessibility (calculated using “Scratch protein predictor” at 30% threshold). Further, the influence of rigidity and conformation of the indole ring (caused by protein secondary structures) on the intrinsic fluorescence properties of proteins were also evaluated using fluorescence of ANSHSA complexes, CD spectroscopy as well as with trypsin digestion experiments. The outcomes obtained clearly demonstrated GnHCl preferably disrupt helix as compared to the beta β-sheets whereas, urea found was more effective in disrupting β-sheets as compared to the helices. The other way round the proteins which have shown detectable change in the intrinsic fluorescence at lower concentrations of GnHCl were rich in helices whereas, the proteins which showed detectable change in the intrinsic fluorescence at lower concentrations of ureawere rich in β-sheets. Since high salt concentrations likeGnHCl and urea interfere in the secondary structure analysis by circular dichroism Spectrometry, the present method of analyzing secondary structures using laser induced autofluorescence will be highly advantageous over existing tools for the same.

Item Type: Article
Uncontrolled Keywords: Autofluorescence; Denaturants; Microenvironment; Urea; Guanidine hydrochloride (GnHCl); Secondary and tertiary structure; Tryptophan (Trp) and tyrosine (Tyr).
Subjects: Life Sciences > MLSC Manipal
Depositing User: KMC Library
Date Deposited: 05 Jun 2017 04:04
Last Modified: 05 Jun 2017 04:04
URI: http://eprints.manipal.edu/id/eprint/148988

Actions (login required)

View Item View Item