Identification of residual structure within denatured antichymotrypsin: implications for serpin folding and misfolding.
The native serpin fold is metastable and possesses the inherent ability to convert into more stable, but inactive, conformations. In order to understand why serpins attain the native fold instead of other more thermodynamically favourable folds we have investigated the presence of residual structure within denatured antichymotrypsin (ACT). Through mutagenesis we created a single tryptophan variant of ACT in which a Trp residue (276) is situated on the H-helix, located within a region known as the B/C barrel. The presence of residual structure around Trp 276 in 5 M guanidine hydrochloride (GdnHCl) was shown by fluorescence and circular dichroism spectroscopy and fluorescence lifetime experiments. The residual structure was disrupted in the presence of 5 M guanidine thiocyanate (GdnSCN). Protein refolding studies showed that significant refolding could be achieved from the GdnHCl denatured state but not the GdnSCN denatured form. The implications of these data on the folding and misfolding of the serpin superfamily are discussed.
Department of Biochemistry and Molecular Biology, Monash University, Vic. 3800, Australia.
Mary C
MC
Pearce
Lisa D
LD
Cabrita
Harvey
H
Rubin
Michael G
MG
Gore
Stephen P
SP
Bottomley
D019791:Guanidine / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry; D015843:Serpins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D000514:alpha 1-Antichymotrypsin / Q000737:chemistry*
D011506:Proteins; D015843:Serpins; D000514:alpha 1-Antichymotrypsin; D014364:Tryptophan; D019791:Guanidine