Sensing the dissociation of a polymeric enzyme by means of an engineered intrinsic probe.
One of the most remarkable characteristics of Brucella lumazine synthase (BLS) is its versatility to undergo reversible dissociation and reassociation as a polymeric scaffold. We have proposed a mechanism of dissociation and unfolding of BLS. Using static light scattering (SLS) analysis, we were able to demonstrate that the decameric assembly dissociates into two different conditions [pH 5 or 2M guanidinium chloride (GdnHCl) pH 7] forming stable folded pentamers. The transition from folded pentamers to unfolded monomers by GdnHCl denaturation is highly cooperative and can be measured by different spectroscopic techniques. In this work, we show the successful insertion of an intrinsic probe to study in more detail the equilibria described in previous publications. For that purpose, we performed single-point mutations of Phe residues 121 and 127, located at the pentamer-pentamer and monomer-monomer interface, respectively, to Trp residues. These mutations produced only a marginal perturbation of the BLS structure. We analyzed the unfolding and stability of the mutants through different techniques: far-and near-UV CD, SLS, dynamic light scattering, and fluorescence spectroscopy. The introduced intrinsic probe could be used to gain insights into the detailed folding and assembly mechanism of this protein.
Fundación Instituto Leloir-IIBBA-CONICET, Av. Patricias Argentinas 435 Buenos Aires (1405), Argentina.
N
N
Ainciart
V
V
Zylberman
P O
PO
Craig
D
D
Nygaard
H R
HR
Bonomi
A A
AA
Cauerhff
F A
FA
Goldbaum
D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002002:Brucella / Q000201:enzymology; D019791:Guanidine; D006863:Hydrogen-Ion Concentration; D008027:Light; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation; D013057:Spectrum Analysis; D013816:Thermodynamics; D014364:Tryptophan
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C044822:6,7-dimethyl-8-ribityllumazine synthase; D014364:Tryptophan; D019791:Guanidine