Experimental and simulative dissociation of dimeric Cu,Zn superoxide dismutase doubly mutated at the intersubunit surface.
The equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase mutant bearing two negative charges in the amino acid clusters at the association interface has been studied, experimentally and computationally, and compared to those of the native enzyme. Pressure-dependent dissociation is observed for the mutant, as observed by the fluorescence shift of the unique tryptophan residue located at the intersubunit surface. The spectral shift occurs slowly, reaching a plateau after 15-20 min, and is fully reversible. Measurement of the degree of dissociation allows us to calculate the standard volume variation upon association and the dissociation constant at atmospheric pressure. On the other hand the native protein is undissociable at any pressure. In the simulative approach, the dissociation free energy has been calculated through the blue moon calculation method for the case of a multidimensional reaction coordinate, corrected for the rotational contribution within the semiclassical approximation for a free rigid-body rotor. The scheme permits to define a definite path for the rupture of the dimer and to calculate the effective force involved in the process. The calculated free energy difference is close to the experimental one, and the value obtained for the mutant is well below that obtained for the native protein, indicating that the theoretical reaction scheme is able to reproduce the experimental trend. Moreover, we find that, when the separation distance increases, the protein structure of the monomer is stable in line with the fast recovery of the original fluorescence properties after decompression, which excludes the presence of partly unfolded intermediates during the dimer-monomer transition.
Maragliano
L
L
INFM-S3 and Department of Physics, University of Modena and Reggio Emilia, Modena, Italy.
Falconi
M
M
Sergi
A
A
Cioni
P
P
Castelli
S
S
Lania
A
A
Stroppolo
M E
ME
Strambini
G
G
Ferrario
M
M
Desideri
A
A
D000596:Amino Acids / Q000737:chemistry; D001703:Biophysics / Q000379:methods*; D019281:Dimerization; D046911:Macromolecular Substances / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D015233:Models, Statistical; D009154:Mutation; D010776:Photobacterium / Q000201:enzymology; D011312:Pressure; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012984:Software; D013482:Superoxide Dismutase / Q000737:chemistry*; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D000596:Amino Acids; D046911:Macromolecular Substances; D011506:Proteins; D011994:Recombinant Proteins; D014364:Tryptophan; D013482:Superoxide Dismutase