The length of a single turn controls the overall folding rate of "three-fingered" snake toxins.
Snake curaremimetic toxins are short all-beta proteins, containing several disulfide bonds which largely contribute to their stability. The four disulfides present in snake toxins make a "disulfide beta-cross"-fold that was suggested to be a good protein folding template. Previous studies on the refolding of snake toxins (Ménez, A. et al. (1980) Biochemistry 19, 4166-4172) showed that this set of natural homologous proteins displays different rates of refolding. These studies suggested that the observed different rates could be correlated to the length of turn 2, one out of five turns present in the toxins structure and close to the "disulfide beta-cross". To demonstrate this hypothesis, we studied the refolding pathways and kinetics of two natural isotoxins, toxin alpha (Naja nigricollis) and erabutoxin b (Laticauda semifasciata), and two synthetic homologues, the alpha mutants, alpha60 and alpha62. These mutants were designed to probe the peculiar role of the turn 2 on the refolding process by deletion or insertion of one residue in the turn length that reproduced the natural heterogeneity at that locus. The refolding was studied by electrospray mass spectrometry (ESMS) time-course analysis. This analysis permitted both the identification and quantitation of the population of intermediates present during the process. All toxins were shown to share the same sequential scheme for disulfide bond formation despite large differences in their refolding rates. The results presented here demonstrate definitely that no residues except those forming turn 2 accounted for the observed differences in the refolding rate of toxins.
Ruoppolo
M
M
Dipartimento di Chimica, Università degli Studi di Salerno, Italy.
Moutiez
M
M
Mazzeo
M F
MF
Pucci
P
P
Ménez
A
A
Marino
G
G
Quéméneur
E
E
D000478:Alkylation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D003039:Cobra Neurotoxin Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D004870:Erabutoxins / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D017510:Protein Folding; D017433:Protein Structure, Secondary
D003039:Cobra Neurotoxin Proteins; D004870:Erabutoxins; C025154:alpha-cobratoxin; C032917:erabutoxin b