title: Tryptophan dynamics in the exploration of micro-conformational changes of refolded β-lactoglobulin after thermal exposure: a steady state and time-resolved fluorescence approach.
abstract: Refolding intermediates of proteins, including molten globules, are likely to undergo dynamic conformational transitions. In this work, thermal unfolding and refolding of bovine β-lactoglobulin (β-lg) have been revisited to encounter such intermediate states. Lower thermal range (< 80°C) was selected to avoid irreversible aggregate formation. The gross kinetic refolding as monitored with the fluorophore, Trp19, was likely to be reversible but alteration in time resolved fluorescence parameters ruled out the possibility of micro-structural reversibility for the refolded partner. Time resolved fluorescence showed that the refolded protein still lacks some intact native conformation. Far-UV CD signals lack the signature of any secondary structural distortion in global structural context whereas near-UV CD signals were strongly indicative of perturbation in micro-structure surrounding the aromatic moieties which hardly revives after cooling. Steady state anisotropy results showed successfully the break-down of dimer to monomer form of β-lg within 50°C temperature range and augmentation in anisotropy up on further thermal stress reflected the reorganization of tryptophan residues into more restricted and rigid micro-environment as well as irreversible disulfide-linked dimer formation. Reliability of conformational reversibility in the thermal unfolding-refolding is still enigmatic on micro and global structural perspectives. Intermediate state prior to the completion of refolding of thermally exposed β-lg was identified through fluorescence studies.
authors.affiliation: Organic Chemistry Section, Department of Chemistry, Jadavpur University, 188, Raja S.C. Mullik Road, Jadavpur, Kolkata 700 032, West Bengal, India. uhalder2002@yahoo.com
authors.forename: Umesh C
authors.initials: UC
authors.lastname: Halder
authors.forename: Jishnu
authors.initials: J
authors.lastname: Chakraborty
authors.forename: Niloy
authors.initials: N
authors.lastname: Das
authors.forename: Sayantan
authors.initials: S
authors.lastname: Bose
mesh_terms: D000818:Animals; D002417:Cattle; D005454:Fluorescence Polarization; D006358:Hot Temperature; D007782:Lactoglobulins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D058849:Protein Refolding; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014364:Tryptophan
chemical_list: D007782:Lactoglobulins; D014364:Tryptophan