High affinity binding of Bcl-xL to cytochrome c: possible relevance for interception of translocated cytochrome c in apoptosis.

The release of cytochrome c from mitochondria and apoptosis relies on several preferential and selective interactions involving the Bcl-2 family of proteins. There is, however, no direct evidence for the interaction of cytochrome c with these proteins at any stage of apoptosis. To investigate if any pro-survival protein from the Bcl-2 family could intercept cytochrome c after its translocation from mitochondria, the interaction of cytochrome c with bacterially expressed human Bcl-x(L) was studied at pH 7. In size-exclusion chromatography, purified full-length His(6)-tagged Bcl-x(L) migrated as both dimer and monomer, of which the monomeric fractions were used for experiments. Coimmunoprecipitation studies show that cytochrome c interacts with Bcl-x(L). The extent of caspase activity in cell lysate elicited by externally added cytochrome c is reduced when a preincubated mixture of Bcl-x(L) and cytochrome c is used instead. Equilibrium binding monitored by optical absorption of cytochrome c as a function of titrating concentrations of Bcl-x(L) yields the association constant, K(ass) = 8.4(+/- 4) x 10(6) M(-1) (binding affinity, K(diss) = 1/K(ass) approximately 120 nM) which decreases at high ionic strength. The rates for binding of Bcl-x(L) to cytochrome c, studied by stopped-flow kinetics at pH 7, show that the bimolecular rate constant for binding, k(bi) = 0.24 x 10(6) M(-1) s(-1). Values of the thermodynamic and kinetic parameters for Bcl-x(L)-cytochrome c interaction are very similar to those known for regulatory protein-protein interactions in apoptosis.

Glassy dynamics in the folding landscape of cytochrome c detected by laser photolysis.

Ferrocytochrome c liganded with CO refolds to a nativelike compact state, called the M state, where the non-native Fe2+-CO contact persists. The M state resembles the generic molten globule-like states and can be driven to the native state by pulsed laser photolysis of the CO ligand. The microsecond kinetics measured all across the guanidine hydrochloride unfolding transition of the protein produce a chevron plot with accentuated rollover in the folding limb, suggesting a glass transition of M en route to N. The rate of exit of the folding structure from the kinetic trap(s) limits the overall rate of folding of M to N. Sulfate-induced deceleration of the observed folding rate suggests that the folding structure indeed is transiently frozen in glassy traps. The results connect the post-transition features of the funnel paradigm.

The alkali molten globule state of ferrocytochrome c: extraordinary stability, persistent structure, and constrained overall dynamics.

This paper describes the structural and dynamic properties of a hitherto uncovered alkali molten globule (MG) state of horse "ferrocytochrome c" (ferrocyt c). Several experimental difficulties mainly because of heme autoxidation and extraordinary stability of ferrocyt c have been overcome by working with the carbonmonoxide-bound molecule under extremely basic condition (pH 13) in a strictly anaerobic atmosphere. Structural and molecular properties extracted from basic spectroscopic experiments suggest that cations drive the base-denatured CO-liganded protein to the MG state. The stability of this state is approximately 5.2 kcal mol(-)(1), and the guanidinium-induced unfolding transition is sharp (m(g) approximately 2.3 kcal mol(-)(1) M(-)(1)), suggesting contents of rigid tertiary structure. Strategic experiments involving the measurement of the CO association rate to the base-denatured protein and intrachain diffusion rates measured by laser photolysis of CO indicate a substantially restricted overall motion and stiffness of the polypeptide chain in the MG state. Possible placement of the state in the folding coordinate of ferrocyt c is discussed.

Protein stiffening and entropic stabilization in the subdenaturing limit of guanidine hydrochloride.

Subdenaturing concentrations of guanidine hydrochloride (GdnHCl) stabilize proteins. For ferrocytochrome c the stabilization is detected at subglobal level with no measured change in global stability. These deductions are made by comparing observed rates of thermally driven ferrocytochrome cHCO reactions with global unfolding rates of ferrocytochrome c measured by stopped flow and NMR hydrogen exchange in the presence of a wide range of GdnHCl concentrations at pH 7, 22 degrees C.