Reconstitution of Acid-Denatured Holomyoglobin Studied by Time-Resolved Electrospray Ionization Mass Spectrometry

Abstract

Time-resolved electrospray ionization (ESI) mass spectrometry (MS) is a new technique for studying the kinetics of protein folding reactions. It can monitor both changes in the protein conformation and the loss or binding of protein ligands as a function of time. Time-resolved ESI MS was previously used to monitor the acid-induced unfolding of holomyoglobin (hMb). The native form of this protein is characterized by a tightly folded conformation and a heme group that is noncovalently attached to the protein. Acid-induced denaturation induces substantial unfolding of the polypeptide chain and disruption of the heme-protein interactions. In this work, time-resolved ESI MS is used to study the reverse reaction, i.e., reconstitution of acid-denatured hMb. To examine the mechanism and the kinetics of this reaction, a continuous-flow setup with two sequential mixing steps was developed. The data presented in this work show that reconstitution involves the formation of various short-lived intermediates such as tightly folded myoglobin without a heme group and several nativelike forms of the protein that are bound to more than one heme. The occurrence of these transient states is most likely due to the rapid aggregation of free heme in solution.

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

“…Close inspection reveals the presence of a third species, which corresponds to Mb with two heme groups attached. Previous studies strongly suggest that these ions reflect the presence of solution-phase proteins that are bound to heme dimers [39,51]. The deconvoluted mass distribution in Figure 2b shows three major peaks, corresponding to aMb (16950 Da), hMb, and Mb with two heme groups (18181 Da).…”

“…Qualitatively, the heme dispersion profile resembles that shown in Figure 3a, which was calculated based on the assumption of no diffusion at all (note the signal onset around t ϭ l/v max Ϸ 940 s, in Figures 3a and 6b). We ascribe this effect to the known tendency of heme to undergo extensive aggregation at acidic pH (see [39,51] and references therein). Presumably, the Stokes radii of these aggregates are large enough to substantially suppress diffusion on the experimental time scale of ϳ2500 s. The observation of a totally different diffusion behavior for heme and protein clearly indicates that noncovalent interactions between the two species are extremely weak or nonexistent for these acidic conditions.…”

“…The same pH was used for the ESI-MS experiments described below. In the absence of organic cosolvents, the protein adopts a tightly folded structure at pH 10, with the heme group seated in the binding pocket (see [39,51] and references therein).…”

“…These considerations imply that the presence or absence of noncovalent complex ions in an ESI mass spectrum does not necessarily allow conclusions to be drawn regarding specific solution phase interactions. Control experiments are often required to rule out false-positive or false-negative scenarios [24,38,39]. It appears that these issues are at least partly responsible for the limited acceptance of ESI-MS as a general tool for "mix and measure" experiments in the context of high-throughput screening tests [3].…”

Collisional cooling enhances the ability to observe non-covalent interactions within the inducible nitric oxide synthase oxygenase domain: Dimerization, complexation, and dissociation

“…[45] to behave in this manner (i.e., bind two heme molecules per monomer) in the gas phase. Deconvolution of spectra obtained at low orifice potentials (30 V or less) reveals monomers with an average mass of 51245 Ϯ 3 Da, and dimers with an average mass of 103174 Ϯ 6 Da.…”