title: Identification of protein secondary structures by laser induced autofluorescence: A study of urea and GnHCl induced protein denaturation.
abstract: In the present study an attempt has been made to interrogate the bulk secondary structures of some selected 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 (3M, 6M, 9M) and GnHCl (2M, 4M, 6M) and the corresponding steady state autofluorescence spectra were recorded at 281nm 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 ANS-HSA 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 urea were rich in β-sheets. Since high salt concentrations like GnHCl 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.
authors.affiliation: Department of Biophysics, School of Life Sciences, Manipal University, Manipal, Karnataka 576104, India.
authors.forename: Manjunath
authors.initials: M
authors.lastname: Siddaramaiah
authors.affiliation: School of Life Sciences, Manipal University, Manipal, Karnataka 576104, India.
authors.forename: Kapaettu
authors.initials: K
authors.lastname: Satyamoorthy
authors.affiliation: Department of Radiation Biology and Toxicology, School of Life Sciences, Manipal University, Manipal, Karnataka 576104, India.
authors.forename: Bola Sadashiva Satish
authors.initials: BSS
authors.lastname: Rao
authors.affiliation: School of Life Sciences, Manipal University, Manipal, Karnataka 576104, India.
authors.forename: Suparna
authors.initials: S
authors.lastname: Roy
authors.affiliation: Department of Biophysics, School of Life Sciences, Manipal University, Manipal, Karnataka 576104, India.
authors.forename: Subhash
authors.initials: S
authors.lastname: Chandra
authors.affiliation: Department of Biophysics, School of Life Sciences, Manipal University, Manipal, Karnataka 576104, India. Electronic address: kkmahato@gmail.com.
authors.forename: Krishna Kishore
authors.initials: KK
authors.lastname: Mahato
mesh_terms: D000818:Animals; D002417:Cattle; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology*; D006801:Humans; D007834:Lasers; D011489:Protein Denaturation / Q000187:drug effects*; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology*
chemical_list: D011506:Proteins; D014508:Urea; D019791:Guanidine
keywords: Autofluorescence; Denaturants; Guanidine hydrochloride (GnHCl); Microenvironment; Secondary and tertiary structure; Tryptophan (Trp) and tyrosine (Tyr); Urea