PUMA
Istituto di Biofisica     
Cioni P., Pesce A., Morozzo Della Rocca B., Castelli S., Falconi M., Parrilli L., Bolognesi M., Strambini G. B., Desideri A. Active-site Copper and Zinc Ions Modulate the Quaternary Structure of Prokaryotic Cu,Zn Superoxide Dismutase. In: Journal of Molecular Biology, vol. 326 (5) pp. 1351 - 1360. Elsevier, 2003.
 
 
Abstract
(English)
The influence of the constitutive metal previous termionsnext term on the equilibrium properties of dimeric Photobacterium leiognathi previous termCu,Zn superoxide dismutasenext term has been studied for the wild-type and for two mutant protein forms bearing a negative charge in the amino acid clusters at the dimer association interface. Depletion of previous termcopper and zincnext term dissociates the two mutant proteins into monomers, which reassemble toward the dimeric state upon addition of stoichiometric amounts of previous termzinc.next term Pressure-dependent dissociation is observed for the previous termcoppernext term-depleted wild-type and mutated enzymes, as monitored by the fluorescence shift of a unique tryptophan residue located at the subunit association interface. The spectral shift occurs slowly, reaching a plateau after 15-20 minutes, and is fully reversible. The recovery of the original fluorescence properties, after decompression, is fast (less than four minutes), suggesting that the isolated subunit has a relatively stable previous termstructure,next term and excluding the presence of stable intermediates during the dimer-monomer transition. The dimer dissociation process is still incomplete at 6.5 kbar for the previous termcoppernext term-depleted wild-type and mutated enzymes, at variance with what is generally observed for oligomeric proteins that dissociate below 3 kbar. Measurement of the degree of dissociation, at two different protein concentrations, allows us to calculate the standard volume variation upon association, ΔV, and the dissociation constant Kd0, at atmospheric pressure, (25 ml/mol and 310−7 M, respectively). The holoprotein is fully dimeric even at 6.5 kbar, which allows us to evaluate a lower ΔG limit of 11.5 kcal/mol, corresponding to a dissociation constant Kd0<10−9 M.
URL: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WK7-47XNNH2-7&_user=3967543&_coverDate=03%2F07%2F2003&_alid=807248012&_rdoc=1&_fmt=high&_orig=search&_cdi=6899&_sort=d&_docanchor=&view=c&_ct=1&_acct=C000061181&_version=1&_urlVersion=0&_userid=39
DOI: 10.1016/S0022-2836(03)00047-0
Subject Superoxide dismutase
Subunit interaction
Quaternary structure
Pressure dissociation
Fluorescence spectroscopy


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