The conformational stability and thermodynamics of Fur A (ferric uptake regulator) from Anabaena sp. PCC 7119.
Fur (ferric uptake regulator) is a key bacterial protein that regulates iron acquisition and its storage, and modulates the expression of genes involved in the response to different environmental stresses. Although the protein is involved in several regulation mechanisms, and members of the Fur family have been identified in pathogen organisms, the stability and thermodynamic characterization of a Fur protein have not been described. In this work, the stability, thermodynamics and structure of the functional dimeric Fur A from Anabaena sp. PCC 7119 were studied by using computational methods and different biophysical techniques, namely, circular dichroism, fluorescence, Fourier-transform infrared, and nuclear magnetic resonance spectroscopies. The structure, as monitored by circular dichroism and Fourier-transform infrared, was composed of a 40% of alpha-helix. Chemical-denaturation experiments indicated that Fur A folded via a two-state mechanism, but its conformational stability was small with a value of DeltaG = 5.3 +/- 0.3 kcal mol(-1) at 298 K. Conversely, Fur A was thermally a highly stable protein. The high melting temperature (Tm = 352 +/- 5 K), despite its moderate conformational stability, can be ascribed to its low heat capacity change upon unfolding, DeltaCp, which had a value of 0.8 +/- 0.1 kcal mol(-1) K(-1). This small value is probably due to burial of polar residues in the Fur A structure. This feature can be used for the design of mutants of Fur A with impaired DNA-binding properties.
Hernández
José A
JA
Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain.
Meier
Jörg
J
Barrera
Francisco N
FN
de los Paños
Olga Ruiz
OR
Hurtado-Gómez
Estefanía
E
Bes
M Teresa
MT
Fillat
María F
MF
Peleato
M Luisa
ML
Cavasotto
Claudio N
CN
Neira
José L
JL
D017033:Anabaena / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry*; D003198:Computer Simulation; D004355:Drug Stability; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012097:Repressor Proteins / Q000032:analysis* / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics
D001426:Bacterial Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial