Organization and dynamics of tryptophan residues in tetrameric and monomeric soybean agglutinin: studies by steady-state and time-resolved fluorescence, phosphorescence and chemical modification.
We have investigated the organization and dynamics of tryptophan residues in tetrameric, monomeric and unfolded states of soybean agglutinin (SBA) by selective chemical modification, steady-state and time-resolved fluorescence, and phosphorescence. Oxidation with N-bromosuccinimide (NBS) modifies two tryptophans (Trp 60 and Trp 132) in tetramer, four (Trp 8, Trp 203 and previous two) in monomer, and all six (Trp 8, Trp 60, Trp 132, Trp 154, Trp 203 and Trp 226) in unfolded state. Utilizing wavelength-selective fluorescence approach, we have observed a red-edge excitation shift (REES) of 10 and 5 nm for tetramer and monomer, respectively. A more pronounced REES (21 nm) is observed after NBS oxidation. These results are supported by fluorescence anisotropy experiments. Acrylamide quenching shows the Stern-Volmer constant (K(SV)) for tetramer, monomer and unfolded SBA being 2.2, 5.0 and 14.6 M(-1), respectively. Time-resolved fluorescence studies exhibit biexponential decay with the mean lifetime increasing along tetramer (1.0 ns) to monomer (1.9 ns) to unfolded (3.6 ns). Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 408.6 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 411.8 and 407.2 nm for monomer and unfolded SBA, respectively. The exposure of hydrophobic surface in SBA monomer has been examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, which shows approximately 20-fold increase in ANS fluorescence compared to that for tetramer. The mean lifetime of ANS also shows a large increase (12.0 ns) upon binding to monomer. These results may provide important insight into the role of tryptophans in the folding and association of SBA, and oligomeric proteins in general.
Molla
Anisur R
AR
Department of Chemistry & Biochemistry, Presidency College, 86/1 College Street, Kolkata 700 073, West Bengal, India.
Maity
Shyam S
SS
Ghosh
Sanjib
S
Mandal
Dipak K
DK
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001974:Bromosuccinimide / Q000737:chemistry; D005454:Fluorescence Polarization; D010084:Oxidation-Reduction; D037121:Plant Lectins / Q000737:chemistry*; D011487:Protein Conformation; D055503:Protein Multimerization; D030262:Soybean Proteins / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*; D010835:Phytohemagglutinins
D000817:Anilino Naphthalenesulfonates; D001974:Bromosuccinimide; D037121:Plant Lectins; D030262:Soybean Proteins; D014364:Tryptophan; C515594:8-anilino-1-naphthalenesulfonic acid; C028824:soybean lectin; D010835:Phytohemagglutinins