Folding mechanism of Pseudomonas aeruginosa cytochrome c551: role of electrostatic interactions on the hydrophobic collapse and transition state properties.
We report on the folding kinetics of the small 82 residue cytochrome c551from Pseudomonas aeruginosa. The presence of two Trp residues (Trp56 and Trp77) allows the monitoring of fluorescence quenching on refolding in two different regions of the protein. A single His residue (the iron-coordinating His16) permits the study of refolding in the absence of miscoordination events. After identification of the kinetic traps (Pro isomerization and aggregation of denatured protein), overall refolding kinetics is described by two processes: (i) a burstphase collapse (faster than milliseconds) which we show to be a global event leading to a state whose compactness depends on the overall net charge; at the isoeletric pH (4.7), it is maximally compact, while above and below it is more expanded; and (ii) an exponential phase (in the millisecond time range) leading to the native protein via a transition state(s) possibly involving the formation of a specific salt bridge between Lys10 and Glu70, at the contact between the N and C-terminal helices. Comparison with the widely studied horse cytochrome c allows the discussion of similarities and differences in the folding of two proteins which have the same "fold" despite a very low degree of sequence homology (<30 %).
Travaglini-Allocatelli
C
C
Istituto Pasteur-Fondazione Cenci Bolognetti and Centro di Biologia Molecolare del CNR Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Università di Roma "La Sapienza", Piazzale A. Moro 5, Rome, I00185, Italy.
Cutruzzolà
F
F
Bigotti
M G
MG
Staniforth
R A
RA
Brunori
M
M
D001426:Bacterial Proteins; D003574:Cytochrome c Group / Q000737:chemistry*; D018698:Glutamic Acid; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D055672:Static Electricity
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D018698:Glutamic Acid; C017167:cytochrome C(551); D008239:Lysine