Probing local secondary structure by fluorescence: time-resolved and circular dichroism studies of highly purified neurotoxins.
The relationship between beta-sheet secondary structure and intrinsic tryptophan fluorescence parameters of erabutoxin b, alpha-cobratoxin, and alpha-bungarotoxin were examined. Nuclear magnetic resonance and x-ray crystallography have shown that these neurotoxins have comparable beta-sheet, beta-turn, and random coil secondary structures. Each toxin contains a single tryptophan (Trp) residue within its beta-sheet. The time-resolved fluorescence properties of native erabutoxin b and alpha-cobratoxin are best described by triple exponential decay kinetics, whereas native alpha-bungarotoxin exhibits more than four lifetimes. The disulphide bonds of each toxin were reduced to facilitate carboxymethylation and amidocarboxymethylation. The two different toxin derivatives of all three neurotoxins displayed triple exponential decay kinetics and were completely denatured as evidenced by circular dichroism (random coil). The concentration (c) values of the three fluorescence decay times (time-resolved fluorescence spectroscopy (TRFS)) were dramatically different from those of the native toxins. Each neurotoxin, treated with different concentrations of guanidinium hydrochloride (GuHCl), was studied both by circular dichroism and TRFS. Disappearance of the beta-sheet secondary structural features with increasing concentrations of GuHCl was accompanied by a shift in the relative contribution (c value) of each fluorescence decay time (TRFS). It was found that certain disulphide residues confer added stability to the beta-sheet secondary structure of these neurotoxins and that the center of the beta-sheet is last to unfold. These titrations show that Trp can be used as a very localized probe of secondary structure.
Department of Biochemistry, University of Ottawa, Ontario, Canada.
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Dahms
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Szabo
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002038:Bungarotoxins / Q000737:chemistry; D002942:Circular Dichroism; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry; D004870:Erabutoxins / Q000737:chemistry; D019791:Guanidine; D006146:Guanidines; D066298:In Vitro Techniques; D008958:Models, Molecular; D009498:Neurotoxins / Q000737:chemistry* / Q000302:isolation & purification; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D002038:Bungarotoxins; D003039:Cobra Neurotoxin Proteins; D006146:Guanidines; D009498:Neurotoxins; D004870:Erabutoxins; C025154:alpha-cobratoxin; D014364:Tryptophan; C032917:erabutoxin b; D019791:Guanidine