title: Localization and environment of tryptophans in different structural states of concanavalin A.
abstract: We have investigated the localization and environment of tryptophan residues in different quaternary and conformational states (tetrameric, dimeric, monomeric and unfolded) of metallized and demetallized concanavalin A (ConA) by selective chemical modification, fluorescence, and phosphorescence. ConA has four tryptophan residues (Trp 40, Trp 88, Trp 109 and Trp 182) per subunit. The pattern of oxidation by N-bromosuccinimide (NBS) shows that NBS modifies, in dimer, only Trp 182 which remains inaccessible in tetramer, two (Trp 88 along with Trp 182) in monomer, all four in unfolded form in presence of EDTA, and three (possibly Trp 40 along with Trp 88 and Trp 182) in unfolded form from native or remetallized ConA. Utilizing wavelength-selective fluorescence approach, we have observed a red edge excitation shift (REES) of 6-8 nm for tetramer and dimer. A more pronounced REES (11 nm) is observed for oxidized monomer compared to REES (3 nm) for unoxidized species. Acrylamide quenching shows the Stern-Volmer constant (K(SV)) for dimer, monomer, unfolded ConA and unfolded apo-ConA being 3.8, 5.2, 12.8, 14.0 M(-1), respectively. Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 406.2 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 413.2 for dimer and 412.6 nm for monomer, while the (0,0) band of the oxidized monomer is red shifted to 414.4 nm. These results may provide important insight into subtlety of organization and environment of tryptophans in the context of folding and structural studies of oligomeric proteins including lectins.
authors: Department of Chemistry & Biochemistry, Presidency University, Kolkata 700 073, India.
authors: Pritha
authors: P
authors: Mandal
authors: Dipak K
authors: DK
authors: Mandal
mesh_terms: D003208:Concanavalin A / Q000737:chemistry*; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D003208:Concanavalin A; D014364:Tryptophan