High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies

Rose, Rebecca; Damoc, Eugen; Denisov, Eduard; Makarov, Alexander; Heck, Albert J. R.

doi:10.1038/nmeth.2208

Cited by 365 publications

(484 citation statements)

References 23 publications

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“…The Functional Dimer Interface of bGP-We found that the association of two GP monomers into the functional dimer involves helix 2 (residues 49 -77), the ␤4/␤5 loop (residues 180 -198), the capЈ loop (residues [41][42][43][44][45][46][47][48], and the tower helices (residues 259 -278), as observed for mGP and lGP (Fig. 5, a and b).…”

Section: Resultsmentioning

confidence: 59%

“…Native MS was performed on samples containing bGP (1 mg/ml) and phosphorylated bGP (1 mg/ml) using an Exactive Plus EMR mass spectrometer (Thermo Fisher Scientific), which allows analysis of proteins and complexes in native-like states (41). The proteins were desalted by buffer exchange prior to mass spectrometry analysis.…”

Section: Purification Of Recombinant Bgp From E Coli-extractionmentioning

confidence: 99%

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Insights into Brain Glycogen Metabolism

Mathieu

Sierra-Gallay

Duval

et al. 2016

Journal of Biological Chemistry

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Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 Å ) and in complex with its allosteric activator AMP (3.4 Å ). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen.

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Section: Resultsmentioning

confidence: 59%

Section: Purification Of Recombinant Bgp From E Coli-extractionmentioning

confidence: 99%

Insights into Brain Glycogen Metabolism

Mathieu

Sierra-Gallay

Duval

et al. 2016

Journal of Biological Chemistry

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“…The experiments described here were carried out using a quadrupoletime-of-flight (Q-TOF) high-mass type of mass spectrometer that has lower resolution in comparison to the Orbitrap instrument (Thermo) (12). MS conditions were therefore first optimized to achieve highly resolved peaks reflecting the small differences in mass between apo-GroEL (801,312 Da) and its nucleotide-bound states (Table S1).…”

Section: Resultsmentioning

confidence: 99%

Allosteric mechanisms can be distinguished using structural mass spectrometry

Dyachenko

Gruber

Shimon³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

133

151

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The activity of many proteins, including metabolic enzymes, molecular machines, and ion channels, is often regulated by conformational changes that are induced or stabilized by ligand binding. In cases of multimeric proteins, such allosteric regulation has often been described by the concerted Monod-Wyman-Changeux and sequential Koshland-Némethy-Filmer classic models of cooperativity. Despite the important functional implications of the mechanism of cooperativity, it has been impossible in many cases to distinguish between these various allosteric models using ensemble measurements of ligand binding in bulk protein solutions. Here, we demonstrate that structural MS offers a way to break this impasse by providing the full distribution of ligand-bound states of a protein complex. Given this distribution, it is possible to determine all the binding constants of a ligand to a highly multimeric cooperative system, and thereby infer its allosteric mechanism. Our approach to the dissection of allosteric mechanisms relies on advances in MSwhich provide the required resolution of ligand-bound states-and in data analysis. We validated our approach using the well-characterized Escherichia coli chaperone GroEL, a double-heptameric ring containing 14 ATP binding sites, which has become a paradigm for molecular machines. The values of the 14 binding constants of ATP to GroEL were determined, and the ATP-loading pathway of the chaperone was characterized. The methodology and analyses presented here are directly applicable to numerous other cooperative systems and are therefore expected to promote further research on allosteric systems.chaperonins | Hill coefficient M ultimeric proteins are often subject to allosteric regulation that is achieved by conformational changes induced or stabilized by ligand binding (1). Such allosteric regulation has been described by two classic models: (i) the Monod-WymanChangeux (MWC) model (2), in which conformational changes occur in a concerted manner and symmetry is conserved, and (ii) the Koshland-Némethy-Filmer (KNF) model (3), in which conformational changes take place in a sequential manner and symmetry is broken. In addition, it has been proposed more recently that conformational changes can take place in a probabilistic manner (4). The allosteric control of protein activity is frequently manifested in sigmoidal plots of initial reaction velocity or fractional saturation as a function of the ligand (substrate) concentration that indicates positive cooperativity in ligand binding. It has been impossible, however, to extract any mechanistic insights from these plots (5) because they only show how an average property of the ensemble (e.g., fractional saturation) changes with ligand concentration and do not reveal how the distribution of ligand-bound states changes with ligand concentration. Thus, for example, it is not possible to determine from such sigmoidal plots whether an allosteric transition takes place in a concerted MWC-like fashion (2) or via a sequential KNF-like mechanism (3). T...

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“…Indeed, the AMP-PNP density shows a well-defined density segment (Fig. 3B), for which the fitted atomic model suggests that it corresponds to the very N-terminal residues (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. These N-terminal 20 residues are absent from all p97 crystal structures but one protomer of the ATPcS-bound IBMPFD mutant A232E, which includes residues 12-20 [10].…”

Section: Conformational Changes Of the N-d1 Segmentmentioning

confidence: 99%

Nucleotide‐dependent conformational changes of the AAA+ ATPase p97 revisited

et al. 2016

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Edited by Peter BrzezinskiThe ubiquitous AAA-ATPase p97 segregates ubiquitylated proteins from their molecular environment. Previous studies of the nucleotide-dependent conformational changes of p97 were inconclusive. Here, we determined its structure in the presence of ADP, AMP-PNP, or ATP-cS at 6.1-7.4 A resolution using single particle cryo-electron microscopy. Both AAA domains, D1 and D2, assemble into essentially six-fold symmetrical rings. The pore of the D1-ring remains essentially closed under all nucleotide conditions, whereas the D2-ring shows an iris-like opening for ADP. The largest conformational changes of p97 are 'swinging motions' of the N-terminal domains, which may enable segregation of ubiquitylated substrates from their environment.

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High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies

Cited by 365 publications

References 23 publications

Insights into Brain Glycogen Metabolism

Insights into Brain Glycogen Metabolism

Allosteric mechanisms can be distinguished using structural mass spectrometry

Nucleotide‐dependent conformational changes of the AAA+ ATPase p97 revisited

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