Alcohol-induced versus anion-induced states of alpha-chymotrypsinogen A at low pH.
Characterization of conformational transition and folding intermediates is central to the study of protein folding. We studied the effect of various alcohols (trifluoroethanol (TFE), butanol, propanol, ethanol and methanol) and salts (K(3)FeCN(6), Na(2)SO(4), KClO(4) and KCl) on the acid-induced state of alpha-chymotrypsinogen A, a predominantly beta-sheet protein, at pH 2.0 by near-UV circular dichroism (CD), far-UV CD and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence measurements. Addition of alcohols led to an increase in ellipticity value at 222 nm indicating the formation of alpha-helical structure. The order of effectiveness of alcohols was shown to be TFE>butanol>propanol>ethanol>methanol. ANS fluorescence data showed a decrease in fluorescence intensity on alcohol addition, suggesting burial of hydrophobic patches. The near-UV CD spectra showed disruption of tertiary structure on alcohol addition. No change in ellipticity was observed on addition of salts at pH 2.0, whereas in the presence of 2 M urea, salts were found to induce a molten globule-like state as evident from the increases in ellipticity at 222 nm and ANS fluorescence indicating exposure of hydrophobic regions of the protein. The effectiveness in inducing the molten globule-like state, i.e. both increase in ellipticity at 222 nm and increase in ANS fluorescence, followed the order K(3)FeCN(6)>Na(2)SO(4)>KClO(4)>KCl. The loss of signal in the near-UV CD spectrum on addition of alcohols indicating disordering of tertiary structure results suggested that the decrease in ANS fluorescence intensity may be attributed to the unfolding of the ANS binding sites. The results imply that the alcohol-induced state had characteristics of an unfolded structure and lies between the molten globule and the unfolded state. Characterization of such partially folded states has important implications for protein folding.
Khan
F
F
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, 202 002, Aligarh, India.
Khan
R H
RH
Muzammil
S
S
D000433:1-Propanol / Q000737:chemistry; D000438:Alcohols / Q000737:chemistry*; D000817:Anilino Naphthalenesulfonates; D002704:Chlorates / Q000737:chemistry; D002919:Chymotrypsinogen / Q000737:chemistry*; D002942:Circular Dichroism; D000431:Ethanol / Q000737:chemistry; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D011189:Potassium Chloride / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012492:Salts / Q000737:chemistry*; D013431:Sulfates / Q000737:chemistry; D014270:Trifluoroethanol / Q000737:chemistry
D000438:Alcohols; D000817:Anilino Naphthalenesulfonates; D002704:Chlorates; D005456:Fluorescent Dyes; D012492:Salts; D013431:Sulfates; C012036:sodium sulfate; D000431:Ethanol; C027132:1-anilino-8-naphthalenesulfonate; D011189:Potassium Chloride; D014270:Trifluoroethanol; D002919:Chymotrypsinogen; D000433:1-Propanol; C022842:chloric acid