The Alkali Molten Globule State of Ferrocytochrome c:  Extraordinary Stability, Persistent Structure, and Constrained Overall Dynamics

Abstract

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.

The Alkali Molten Globule State of Horse Ferricytochrome c: Observation of Cold Denaturation

“…[1][2][3]20,21 Although the complementary cation-stabilized state under extreme basic conditions has been reported for only a few proteins, 22,23,30 the present results for ferricyt c provide growing evidence for the generality of the B state as the cation-stabilized alkali equilibrium state. Since A and B states accumulate under extreme pH conditions, they should be stabilized by the added counter ions through the same mechanism, the solvent ions either reduce the intrapolypeptide electrostatic repulsion or bind directly to the protein charges to form ion pairs, or possibly both.…”

“…In the first set of studies, we used the carbonmonoxy derivative of the reduced form of cyt c (ferrocyt c) at pH 13, and achieved the U B → B transition in the presence of Na + . 30 Those experiments were difficult because ferrocyt c hardly denatures even at the extreme of basic pH, and therefore the CO adduct was used. Here, we present the second set of experiments with ferricyt c where the U B state is directly prepared at pH 13, and the U B → B transition is achieved in the presence of NaCl.…”

On the mechanism of SDS‐induced protein denaturation

“…To understand the mechanism of SDS interaction with unfolded proteins, especially when the latter is highly ionized, changes in the fluorescence intensity of alkaline cyt‐CO was monitored as a function of SDS. Alkaline cyt‐CO is prepared by liganding the heme iron of ferrocyt c with CO at pH 12.75 as described previously 58, 59. The NMR spectrum of alkaline cyt‐CO shows no dispersion of chemical shift 59.…”

“…Alkaline cyt‐CO is prepared by liganding the heme iron of ferrocyt c with CO at pH 12.75 as described previously 58, 59. The NMR spectrum of alkaline cyt‐CO shows no dispersion of chemical shift 59. It is a highly denatured expanded state retaining ∼25% of the native‐state secondary structure, and virtually no trace of tertiary structure.…”

Analysis of the pH-dependent stability and millisecond folding kinetics of horse cytochrome c

“…The decrease in m g -values at alkaline pH relative to neutral pH is likely due to (i) relatively small solvent accessible area in the denatured state of protein at pH ! 11.5, (ii) deviation from two-state folding [95], and (iii) denaturant unfolded cyt c is more compact at alkaline pH than at neutral pH [87,96]. The fluorescence emission intensity of denaturant unfolded cyt c at pH 13 is at least 2e5 fold less relative to pH 7 [87,96], which is possibly due to either quenching by hydroxyl ions or ionization of the phenolic hydroxyl group of tyrosine at pH 13.…”