Equilibrium unfolding of a small bacterial cytochrome, cytochrome c551 from Pseudomonas aeruginosa.
The unfolding of the small cytochrome c551 from the bacterium Pseudomonas aeruginosa has been characterized at equilibrium by circular dichroism (CD) and fluorescence spectroscopy. The process can be described by a two state mechanism and the thermodynamic stability of cytochrome c551 is found to be smaller than that of the larger horse cytochrome c (deltaGw = -8.2 vs. -9.7 kcal/mol); we propose that this finding is related to the absence of an 'omega' loop in the bacterial cytochrome. Cytochrome c551 loses most of its secondary structure at pH 1.5. The acid transition (pKA approximately 2) is highly cooperative (n > or =2); analysis of optical titrations and contact map suggests that (at least) His-16 (proximal Fe3+ ligand) and Glu-70 are both involved in the acid transition. The role of selected hydrophobic, electrostatic and conformational contributions to the overall stability has been investigated by protein engineering. The equilibrium characterization of wild-type and mutant cytochrome c551 supports the view that this small cytochrome is an interesting protein to analyze the thermodynamics and the kinetics of folding in comparison with the widely studied horse cytochrome c.
Bigotti
M G
MG
Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche A. Rossi-Fanelli and Centro di Biologia Molecolare del CNR, Università di Roma La Sapienza, Rome, Italy.
Allocatelli
C T
CT
Staniforth
R A
RA
Arese
M
M
Cutruzzolà
F
F
Brunori
M
M
D000143:Acids / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability / Q000502:physiology; D019791:Guanidine / Q000494:pharmacology; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D012492:Salts / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D000143:Acids; D001426:Bacterial Proteins; D003574:Cytochrome c Group; D012492:Salts; D014508:Urea; C017167:cytochrome C(551); D019791:Guanidine