Conformational changes in azurin from Pseudomona aeruginosa induced through chemical and physical protocols.
Azurin from Pseudomona aeruginosa is a small copper protein with a single tryptophan (Trp) buried in the structure. The Gibbs free energies associated with the folding of holo azurin, calculated monitoring Trp fluorescence and changes in absorbance on the ligand-to-metal band, are different because these techniques probe their local environments, thereby being able to probe different conformational changes. The presence of an intermediate state was observed during the chemical denaturation of the protein. Upon denaturation, a 30-fold increase is observed in the magnitude of the quenching constant of the tryptophan fluorescence by acrylamide, because this residue becomes more accessible to the quencher. Entrapping the protein in sol-gel materials lowers its stability possibly because the solvation properties of the macromolecule are changed. The thermal denaturation of azurin immobilized in a sol-gel monolith is irreversible, which tends to rule out an aggregation mechanism to account for the irreversibility of the denaturation of the protein free in solution. Unlike the Cu(II) ion, the Gd(III) ion accommodates in site B of azurin with high affinity and the folding free energy of Gd-azurin is larger than that of apo azurin.
Fuentes
Lymari
L
Department of Chemistry, University of Puerto Rico, San Juan, Puerto Rico 00931-3346.
Oyola
Jessica
J
Fernández
Mónica
M
Quiñones
Edwin
E
D020106:Acrylamide / Q000737:chemistry; D001400:Azurin / Q000737:chemistry*; D055592:Biophysical Phenomena; D001703:Biophysics; D003300:Copper / Q000737:chemistry; D005682:Gadolinium / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D008024:Ligands; D044367:Phase Transition; D011487:Protein Conformation; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D008024:Ligands; D001400:Azurin; D020106:Acrylamide; D003300:Copper; D014364:Tryptophan; D005682:Gadolinium; D019791:Guanidine