Equilibrium unfolding of proteins monitored by electrospray ionization mass spectrometry: distinguishing two-state from multi-state transitions

Konermann, Lars; Douglas, D. J.

doi:10.1002/(sici)1097-0231(19980430)12:8<435::aid-rcm181>3.0.co;2-f

Cited by 145 publications

(128 citation statements)

References 45 publications

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“…These observations are in line with the well known fact that the ESI charge state distribution represents a sensitive probe of protein conformational changes. It is generally accepted that protein unfolding in solution leads to the formation of more highly charged protein ions in positive-ion ESI [77][78][79][80][81][82][83]. However, the physical basis of this phenomenon is still a matter of debate [23,84,85].…”

Section: Stokes Radiimentioning

confidence: 99%

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

Clark

Konermann

2003

J. Am. Soc. Mass Spectrom.

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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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Section: Stokes Radiimentioning

confidence: 99%

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

Clark

Konermann

2003

J. Am. Soc. Mass Spectrom.

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“…This is because these techniques provide only the population-weight average property under each condition. Electrospray ionization mass spectrometry (ESI-MS), however, has the unique capability of resolving multiple species that are co-populated in solution, provided that these species give rise to different charge state distributions (23)(24)(25)(26). Several models have been proposed to rationalize the observed differences in the charge state distributions of native and nonnative states.…”

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confidence: 99%

Co-populated Conformational Ensembles of β2-Microglobulin Uncovered Quantitatively by Electrospray Ionization Mass Spectrometry

Borysik

Radford²,

Ashcroft

2004

Journal of Biological Chemistry

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Ordered assembly of monomeric human ␤ 2 -microglobulin (␤ 2 m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions (pH <5.0 at 37°C), wild-type ␤ 2 m assembles spontaneously into fibrils with different morphologies. Under these conditions, ␤ 2 m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of ␤ 2 m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9؉ ions. By contrast, under more acidic conditions (pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10؉ and 11؉ ions. The data are reinforced by investigations on two variants of ␤ 2 m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid.␤ 2 -microglobulin (␤ 2 m) 1 is one of a number of proteins known to aggregate into insoluble amyloid deposits in vivo (1-3). These amyloid deposits have been linked to various human maladies such as Alzheimer's disease (4), Huntington's chorea (5), and senile systemic amyloidosis (6). However, for the majority of the ϳ20 known amyloid-related proteins, the structural mechanism of amyloid formation is still unclear, and, for several proteins, the role that different aggregated states play in the manifestation of the disease symptoms remains unresolved (7).␤ 2 m is a small (11,860-dalton) extracellular protein that forms the nonpolymorphic light chain component of the major histocompatibility class I complex. The physiological role of ␤ 2 m has been likened to a chaperone in that it is required for the heavy chain of the major histocompatibility class I complex to fold into a stable secreted entity (8, 9). ␤ 2 m is an all ␤-sheet protein, with a seven-stranded ␤-sandwich structure and a single disulfide bond linking Cys-25 and Cys-80 in the B and F strands (10) (Fig. 1). In healthy individuals, the serum concentration of circulating free ␤ 2 m is ϳ3 mg⅐liter Ϫ1 (11), and the protein is removed from the serum by renal catabolism (12). In individuals undergoing renal dialysis, the serum concentration of ␤ 2 m can rise to...

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“…Mass spectrometry is particularly useful to study protein-ligand interactions, owing to its ability to determine ligand composition and binding stoichiometry by direct mass measurement of the protein-ligand complex following its desorption from solution by means of electrospray ionization (ESI) [1]. Furthermore, charge state distributions in the ESI spectra of folded and unfolded proteins provide a useful tool to study protein conformation at low resolution [4,5] and its relationship to ligand binding [6]. Proteins under native conditions show narrow charge state distributions in mass spectra with a small number of charges, whereas those under denaturing conditions tend to have broader charge state distribution with a higher number of charges.…”

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confidence: 99%

Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS

Kaltashov

Eyles

2003

J. Am. Soc. Mass Spectrom.

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Direct mass spectrometric characterization of interactions between proteins and small hydrophobic ligands often poses a serious problem due to the complex instability in the gas phase. We have developed a method that probes the efficacy of ligand-protein interactions indirectly by monitoring changes in protein flexibility. The latter is assessed quantitatively using a combination of charge state distribution analysis and amide hydrogen exchange under both native and mildly denaturing conditions. The method was used to evaluate binding of a model protein cellular retinoic acid binding protein I to its natural ligand all-trans retinoic acid (RA), isomers 13-cis-and 9-cis-RA, and retinol, yielding the following order of ligand affinities: All-trans RA Ͼ 9-cis RA Ͼ 13-cis RA, with no detectable binding of retinol. This order is in agreement with the results of earlier fluorimetric titration studies. Furthermore, binding energy of the protein to each of retinoic acid isomers was determined based on the measured hydrogen exchange kinetics data acquired under native conditions. (J Am Soc Mass Spectrom 2003, 14, 506 -515)

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Equilibrium unfolding of proteins monitored by electrospray ionization mass spectrometry: distinguishing two-state from multi-state transitions

Cited by 145 publications

References 45 publications

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

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

Co-populated Conformational Ensembles of β2-Microglobulin Uncovered Quantitatively by Electrospray Ionization Mass Spectrometry

Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS

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