Stability and folding/unfolding kinetics of the homotrimeric coiled coil Lpp-56.
The 56 amino acid long protein moiety of the E. coli outer membrane lipoprotein (Lpp-56) contains a 4,3 hydrophobic heptad repeat and forms a parallel, in-register trimeric coiled coil in solution. The unconventional structural properties of Lpp-56 make it an interesting experimental object to study the folding of a trimeric coiled coil. Folding is unusually slow at low temperatures, and the rates of folding and unfolding are balanced in such a way that the thermodynamic equilibrium is established after considerable time at high temperatures or in the presence of denaturants. Here, we examine the stability and the folding/unfolding kinetics of Lpp-56 at neutral pH using GdmCl as the denaturant. Folding and unfolding appear to represent structural transitions between the folded trimer and the unfolded monomer, without detectable intermediates. For the first time, we estimated the unfolding free energy from a direct measurement at equilibrium. Our estimate of DeltaG(U) = 79 +/- 10 kJ mol(-)(1) compares very well with DeltaG(U) approximately 76-88 kJ mol(-)(1) obtained from the kinetic rate constants of refolding (7 x 10(5) M(-)(2) s(-)(2)) and unfolding (10(-)(9) to 10(-)(11) s(-)(1)) and is almost half of the DeltaG(U) recently suggested using a different methodology (Dragan, A. I., Potekhin, S., Sivolob, A., Lu, M., and Privalov, P. L. (2004) Biochemistry 43, 14891-14900). Because GdmCl attenuates electrostatic interactions, the discrepancy can be partly explained by an electrostatic component of the unfolding barrier. The combined information illustrates the difficulties in obtaining a precise biophysical description of proteins that exhibit unusual kinetic properties. Lpp-56 is the first coiled coil for which a high unfolding kinetic barrier has been experimentally demonstrated.
Bjelić
Sasa
S
Biochemisches Institut der Universität Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
Karshikoff
Andrey
A
Jelesarov
Ilian
I
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C099932:Lpp protein, E coli; D019791:Guanidine