Fluoroalcohols induced unfolding of Succinylated Con A: native like beta-structure in partially folded intermediate and alpha-helix in molten globule like state.
Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20-60% (v/v) 2,2,2-trifluoroethanol (TFE) and 2-40% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), Con A at pH 5.0 shows visible aggregation. However, when succinyl Con A was used, no aggregation was observed in the entire concentration range of fluoroalcohols (0-90% v/v TFE and HFIP) and resulted in stable alpha-helix formation. Temperature-induced concentration-dependent aggregation in Con A was also found to be prevented/reduced in succinylated form. Possible role of electrostatic repulsion among residues in the prevention of hydrophobically driven aggregation has been discussed. Results indicate that succinylation of a protein resulted in greater stability (in both beta-sheet and alpha-helical forms) against alcohol-induced and temperature-induced concentration-dependent aggregation and this observation may play significant role in amyloid-forming proteins. Effect of TFE and HFIP on the conformation of a dimeric protein, Succinylated Con A, has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of hydrophobic dye ANS (8-anilinonaphthalene-1-sulfonic acid). Far UV-CD, a probe for secondary structure shows loss of native secondary structure in the presence of low concentration of both the alcohols, TFE (10% v/v) and HFIP (4% v/v). Upon addition of higher concentration of these alcohols, Succinylated Con A exhibited transformation from beta-sheet to alpha-helical structure. Intrinsic tryptophan fluorescence studies, ANS binding and near UV-CD experiments indicate the protein is more expanded, have more exposed hydrophobic surfaces and highly disrupted tertiary structure at 60% (v/v) TFE and 30% (v/v) HFIP concentrations. Taken together, these results it might be concluded that TFE and HFIP induce two intermediate states at their low and high concentrations in Succinyl Con A.
Fatima
Sadaf
S
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
Ahmad
Basir
B
Khan
Rizwan Hasan
RH
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002942:Circular Dichroism; D003208:Concanavalin A / Q000737:chemistry*; D019281:Dimerization; D057927:Hydrophobic and Hydrophilic Interactions; D008856:Microscopy, Fluorescence; D020005:Propanols / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014270:Trifluoroethanol / Q000737:chemistry*; D006845:Hydrocarbons, Fluorinated
D000817:Anilino Naphthalenesulfonates; D003208:Concanavalin A; D020005:Propanols; D014270:Trifluoroethanol; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated; C019558:succinylconcanavalin A; C027132:1-anilino-8-naphthalenesulfonate