Conformational study of spectrin in presence of submolar concentrations of denaturants.
The presence of very low concentrations of the commonly used chemical denaturants, guanidinium chloride (GdmCl) and urea brought about conformational changes in the erythrocyte membrane skeletal protein, spectrin. Evidences in support of changes in the quaternary structure of spectrin have been put forward from quenching study of tryptophan fluorescence, by both steady state and time-resolved measurements, using acrylamide as the quencher. It revealed significant differences between the Stern-Volmer quenching constants (K(SV)) and the fraction of accessible tryptophans (f(e)) observed in absence and presence of GdmCl and urea concentrations below 1 M at which the association of the two subunits remains intact. The steady state anisotropy of both the spectrin tryptophans and the spectrin-bound fluorescence probe, Prodan also indicate changes in the overall flexibility of the spectrin dimer, originating from changes in the quaternary structure of spectrin. Studies on the binding of Prodan, further indicate that conformational changes also occur in spectrin near the Prodan-binding site at the terminal domain of the protein which is reflected in 3-4 fold decrease in the affinity of binding of Prodan to spectrin in the presence of GdmCl and urea compared to that observed in the absence of the denaturants. The dissociation constant (K(d)) of Prodan to spectrin is 0.43 microM at 25 degrees C.
Ray
Sibnath
S
Biophysics Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata, 700064 India.
Bhattacharyya
Malyasri
M
Chakrabarti
Abhijit
A
D015081:2-Naphthylamine / Q000031:analogs & derivatives*; D000818:Animals; D001665:Binding Sites; D019281:Dimerization; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D019791:Guanidine; D066298:In Vitro Techniques; D011487:Protein Conformation; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D012756:Sheep; D013049:Spectrin / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry; D014508:Urea
D005456:Fluorescent Dyes; D013049:Spectrin; C038517:prodan; D014364:Tryptophan; D014508:Urea; D015081:2-Naphthylamine; D019791:Guanidine