Folding of a de novo designed native-like four-helix bundle protein.
The folding of a model native-like dimeric four-helix bundle protein, (alpha(2))(2), was investigated using guanidine hydrochloride, hydrostatic pressure, and low temperature. Unfolding by guanidine hydrochloride followed by circular dichroism and intrinsic fluorescence spectroscopy revealed a highly cooperative transition between the native-like and unfolded states, with free energy of unfolding determined from CD data, DeltaG(unf) = 14.3 +/- 0.8 kcal/mol. However, CD and intrinsic fluorescence data were not superimposable, indicating the presence of an intermediate state during the folding transition. To stabilize the folding intermediate, we used hydrostatic pressure and low temperature. In both cases, dissociation of the dimeric native-like (alpha(2))(2) into folded monomers (alpha(2)) was observed. van't Hoff analysis of the low temperature experiments, assuming a two-state dimer 171-monomer transition, yielded a free energy of dissociation of (alpha(2))(2) of DeltaG(diss) = 11.4 +/- 0.4 kcal/mol, in good agreement with the free energy determined from pressure dissociation experiments (DeltaG(diss) = 10.5 +/- 0.1 kcal/mol). Binding of the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to the pressure- and cold-dissociated states of (alpha(2))(2) indicated the existence of molten-globule monomers. In conclusion, we demonstrate that the folding pathway of (alpha(2))(2) can be described by a three-state transition including a monomeric molten globule-like state.
Chapeaurouge
Alex
A
Departamento de Bioquimica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil. henk@bioqmed.ufrj.br
Johansson
Jonas S
JS
Ferreira
Sérgio T
ST
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D002845:Chromatography; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000494:pharmacology; D019791:Guanidine / Q000737:chemistry; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D011312:Pressure; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D019791:Guanidine