Observation of noncovalent enzyme-substrate and enzyme-product complexes by ion-spray mass spectrometry

Ganem, Bruce; Li, Yu Tsyr; Henion, Jack D.

doi:10.1021/ja00020a085

Cited by 325 publications

(206 citation statements)

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“…Furthermore, the gentle nature of the transfer of proteins from the electrospray droplet to the gas phase means that perturbation of the solution structure of a protein is minimal (39), and there is now significant evidence to suggest that the correlation between the solution and gas phase properties of a protein is significant (27,40,41). An impressive and increasing number of ESI-MS-based approaches now exist with regard to the characterization of the conformational dynamics of proteins (38).…”

Section: Discussionmentioning

confidence: 99%

“…Several models have been proposed to rationalize the observed differences in the charge state distributions of native and nonnative states. Differences in the solvent accessibility of ionizable groups (23,27), heightened Coulomb energies of folded compared with unfolded polyprotonated conformations (28,29), or the increased protection against charge neutralization offered by native protein conformations (30) have all been offered as plausible explanations. Whichever mechanism or combination of processes governs this phenomenon, the net result is that conformational species with more open structures carry, on average, more charges and produce broader ion envelopes centered on higher charge states than their more compact native counterparts (31).…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

mentioning

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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“…Indeed, soon after the introduction of MALDI and ESI, these ionization methods were already used for the study of intact proteins and noncovalent protein complexes (Ganem, Li, & Henion, 1991;Miranker et al, 1993;Mirza, Cohen, & Chait, 1993;. In particular, ESI is well suited to detect and investigate non-covalent complexes by transferring whole intact assemblies into the vacuum inside the mass spectrometer.…”

Section: Introductionmentioning

confidence: 99%

Investigation of intact protein complexes by mass spectrometry

Heck

Heuvel

2004

Mass Spectrometry Reviews

553

554

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I. Introduction 00 II. Electrospray Ionization of Biomacromolecules 00 III. Mass Analyzers for Biomacromolecular Mass Spectrometry 00 A. Collisional Cooling and/or Focusing 00 B. Alternative Mass Analyzers 00 IV. Solution‐Phase Properties of Proteins Complexes 00 A. Protein–Protein Interactions 00 B. Cooperativity and Cofactors 00 C. Dynamics of Assembly 00 V. Gas‐Phase Properties of Protein Complexes 00 A. Tandem Mass Spectrometry 00 B. Charge Separation in Protein Dissociation 00 VI. Future Outlook 00 Acknowledgments 00 References 00 Mass spectrometry has grown in recent years to a well‐accepted and increasingly important complementary technique in structural biology. Especially electrospray ionization mass spectrometry is well suited for the detection of non‐covalent protein complexes and their interactions with DNA, RNA, ligands, and cofactors. Over the last decade, significant advances have been made in the ionization and mass analysis techniques, which makes the investigation of even larger and more heterogeneous intact assemblies feasible. These technological developments have paved the way to study intact non‐covalent protein–protein interactions, assembly and disassembly in real time, subunit exchange, cooperativity effects, and effects of cofactors, allowing us a better understanding of proteins in cellular processes. In this review, we describe some of the latest developments and several highlights. © 2004 Wiley Periodicals, Inc., Mass Spec Rev

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“…This not only avoids the denaturing conditions that have been used previously, but also allows the study of proteins under conditions in which they can be detected as complexes with substrates, products or inhibitors. Initially, ESI MS was restricted to the study of complexes in which the ligand (substrate or product) was covalently bound [1], but increasingly non-covalently bound enzyme complexes are being studied [2][3][4][5]. However, these studies have principally been concerned with the binding of relatively small molecules to enzymes [6], or with protein-protein interactions in natural oligomers, e.g.…”

Section: Introductionmentioning

confidence: 99%

An investigation of the binding of protein proteinase inhibitors to trypsin by electrospray ionization mass spectrometry

Kraunsoe

Aplin

Green³

et al. 1996

FEBS Letters

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The binding of BPTI and SBTI with trypsin has been investigated by ESI MS, using the mutant K15V-BPTI and the chemically modified RcamBPTI as controls. Although high cone voltages (+80 V) produce sharp spectra of BPTI, RcamBPTI, SBTI and trypsin alone, the complexes of BPTI, RcamBPTI and SBTI with trypsin undergo partial dissociation due to collisional activation. At lower cone voltages (+40 V) these non-covalent complexes are stable. The charge distribution on the trypsin and the inhibitors produced by gas phase dissociation of the complexes are markedly different from those of the components alone, indicating that ESI MS provides a novel probe for exploring the ionic interactions at the contact surface of proteins. Moreover, by determining the cone voltage at which the gas phase dissociation of complexes occurs it may be possible to use ESI MS to compare the binding energies of closely related complexes.

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Observation of noncovalent enzyme-substrate and enzyme-product complexes by ion-spray mass spectrometry

Cited by 325 publications

References 2 publications

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

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

Investigation of intact protein complexes by mass spectrometry

An investigation of the binding of protein proteinase inhibitors to trypsin by electrospray ionization mass spectrometry

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