A novel approach for the quantification of ligand-protein interactions is presented. Electrospray ionization mass spectrometry (ESI-MS) is used to monitor the diffusion behavior of noncovalent ligands in the presence of their protein receptors. These data allow the fraction of free ligand in solution to be determined, such that the corresponding dissociation constants can be calculated. A set of conditions is developed that provides an "allowable range" of concentrations for this type of assay. The method is tested by applying it to two different inhibitor-enzyme systems. The dissociation constants measured for benzamidine-trypsin and for N,N',N' '-triacetylchitotriose-lysozyme are (50 +/- 10) and (6 +/- 1) mM, respectively. Both of these results are in good agreement with previous data from the literature. In contrast to traditional ESI-MS-based methods, the approach used in this work does not rely on the preservation of specific solution-type noncovalent interactions in the gas phase. It is shown that this method allows an accurate determination of dissociation constants, even in cases in which the ion abundance ratio of free to ligand-bound protein in ESI-MS does not reflect the corresponding concentration ratio in solution.
The b subunit dimer of the Escherichia coli ATP synthase, along with the ␦ subunit, is thought to act as a stator to hold the ␣ 3  3 hexamer stationary relative to the a subunit as the ␥⑀c 9 -12 complex rotates. Despite their essential nature, the contacts between b and the ␣, , and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b 24 -156 , a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F 1 sector or to complete ATP synthase, or F 1 F 0 -ATPase, utilizes a transmembrane proton gradient to synthesize ATP and is responsible for the final step in oxidative phosphorylation and photophosphorylation. The enzyme (reviewed in Refs. 1-3) is composed of two sectors. The membrane-integral F 0 sector is a proton pore, and in Escherichia coli has a subunit composition of ab 2 c 9 -12 . The membrane-peripheral F 1 sector has a subunit stoichiometry of ␣ 3  3 ␥␦⑀. A key feature of the F 1 sector, as seen in the bovine heart mitochondrial crystal structure (4), is that the ␣ and  subunits alternate in a ring around a lengthy pair of ␣-helices of ␥. Each  subunit bears one catalytic nucleotide-binding site, while non-catalytic nucleotide-binding sites are found on the ␣ subunits. These nucleotide-binding sites are located close to the interfaces between ␣ and  subunits, with one site near each of the six interfaces.Subunits from each sector contribute to the formation of two stalks that join F 1 and F 0 . The ␥ and ⑀ subunits form the central stalk, rotation of which is believed to be caused by translocation of protons across the membrane by the a and c subunits. This rotation is thought to cause conformational changes in the catalytic sites, driving synthesis of ATP (1). It is believed that the ␣ and  subunits are prevented from rotating by a peripheral stalk consisting of ␦ and the two b subunits (5, 6) that joins the ␣ 3  3 complex to the a subunit.In recent years the interaction of the b dimer and ␦ has been well established by a variety of evidence (7-10). The ␦ subunit appears to be located near the crown of the F 1 complex, the part of F 1 furthest from the membrane (11-15). Because b has a single membrane-spanning region at its N terminus, the remainder of the subunit must span a distance of over 100 Å to come in contact with ␦. Consistent with this proposed arrangement, the region of interaction between b and ␦ has been localized to the C terminus of b (10, 16). The hydrophilic domain of b by itself is mostly ␣-helical as measured by circular dichroism (17), and an isolated complex composed of ␦ with the hydrophilic domain of b was demonstrated by sedimentation velocity ultracentrifugation to be highly extended (9). The dimerization domain of b, encompassing residues 53-122, was also shown to be highly extended (18). Therefore the b 2...
This work describes a novel approach for monitoring analyte diffusion in solution that is based on electrospray ionization mass spectrometry (ESI-MS). A mass spectrometer at the end of a laminar flow tube is used to measure the Taylor dispersion of an initially sharp boundary between two solutions of different analyte concentration. This boundary is dispersed by the laminar flow profile in the tube. However, this effect is diminished by analyte diffusion that continuously changes the radial position, and hence the flow velocity of individual analyte molecules. The steepness of the resulting dispersion profile therefore increases with increasing diffusion coefficient of the analyte. A theoretical framework is developed to adapt the equations governing the dispersion process to the case of mass spectrometric detection. This novel technique is applied to determine the diffusion coefficients of choline and cytochrome c. The measured diffusion coefficients, (11.9 +/- 1.0) x 10(-10) m(2) s(-1) and (1.35 +/- 0.08) x 10(-10) m(2) s(-1), respectively, are in agreement with the results of control experiments where the Taylor dispersion of these two analytes was monitored optically. Due to the inherent selectivity and sensitivity of ESI-MS, it appears that the approach described in this work could become a valuable alternative to existing methods for studying diffusion processes, especially for experiments on multicomponent systems.
This study describes a novel approach for monitoring noncovalent interactions in solution by electrospray mass spectrometry (ESI-MS). The technique is based on measurements of analyte diffusion in solution. Diffusion coefficients of a target macromolecule and a potential low molecular weight binding partner are determined by measuring the spread of an initially sharp boundary between two solutions of different concentration in a laminar flow tube (Taylor dispersion), as described in Rapid Commun. Mass Spectrom. 2002Spectrom. , 16, 1454Spectrom. -1462. In the absence of noncovalent interactions, the measured ESI-MS dispersion profiles are expected to show a gradual transition for the macromolecule and a steep transition for the low molecular weight compound. However, if the two analytes form a noncovalent complex in solution the dispersion profiles of the two species will be very similar, since the translational diffusion of the small compound is determined by the slow Brownian motion of the macromolecule. In contrast to conventional ESI-MS-based techniques for studying noncovalent complexes, this approach does not rely on the preservation of solution-phase interactions in the gas phase. On the contrary, "harsh" conditions at the ion source are required to disrupt any potential gasphase interactions between the two species, such that their dispersion profiles can be monitored separately. The viability of this technique is demonstrated in studies on noncovalent heme-protein interactions in myoglobin. Tight noncovalent binding is observed in solutions of pH 10, both in the absence and in the presence of 30% acetonitrile. In contrast, a significant disruption of the noncovalent interactions is seen at an acetonitrile content of 50%. Under these conditions, the diffusion coefficient of heme in the presence of myoglobin is only slightly lower than that of heme in a protein-free solution. A breakdown of the noncovalent interactions is also observed in aqueous solution of pH 2.4, where myoglobin is known to adopt an acid-unfolded conformation. (J Am Soc Mass Spectrom 2003, 14, 430 -441)
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