Protein subunit interactions and structural integrity of amyloidogenic transthyretins: evidence from electrospray mass spectrometry

Nettleton, Ewan J.; Sunde, Margaret; Lai, Zhihong; Kelly, Jeffery W.; Dobson, Christopher M.; Robinson, Carol V.

doi:10.1006/jmbi.1998.1937

Cited by 162 publications

(137 citation statements)

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“…Nanoflow electrospray mass spectra (nESI-MS) were recorded on a Synapt HDMS system and an LCT Premier instrument (Waters Corp.) optimized for the transmission of noncovalent complexes (35). Typically, 3 L solution containing p53 were electrosprayed from gold-coated glass capillaries (36). To preserve the non-covalent interactions in the p53 tetramer, the MS parameters used for the Synapt were: capillary voltage, 1.3-2.0 kV; sample cone, 150 V; trap and transfer collision energy, 100 V; backing pressure 5 mbar, source pressure 6 -7e Ϫ2 mbar; trap and IMS pressure 5e Ϫ2 and 5e Ϫ1 mbar, respectively; time-of-flight analyser pressure 2e Ϫ6 mbar.…”

Section: Methodsmentioning

confidence: 99%

Ultraslow oligomerization equilibria of p53 and its implications

Natan

Hirschberg

Morgner

et al. 2009

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

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The tumor suppressor p53 is in equilibrium at cellular concentrations between dimers and tetramers. Oncogenic mutant p53 (mut) exerts a dominant-negative effect on co-expression of p53 wildtype (wt) and mut alleles in cancer cells. It is believed that wt and mut form hetero-tetramers of attenuated activity, via their tetramerization domains. Using electrospray mass spectrometry on isotopically labeled samples, we measured directly the composition and rates of formation of p53 complexes in the presence and absence of response element DNA. The dissociation of tetramers was unexpectedly very slow (t 1/2 ‫؍‬ 40 min) at 37°C, matched by slow association of dimers, which is approximately four times longer than the half-life of spontaneous denaturation of wt p53. On mixing wt tetramers with the oncogenic contact mutant R273H of low DNA affinity, we observed the same slow formation of only wt 4, wt2mut2, and mut4, in the ratio 1:2:1, on a cellular time scale. On mixing wt and mut with response element DNAs P21 and BAX, we observed only the complexes wt 4.DNA, wt2mut2.DNA, and mut 4.DNA, with relative dissociation constants 1:4:71 and 1:13:85, respectively, accounting for the dominant-negative effect by weakened affinity. p53 dimers assemble rapidly to tetramers on binding to response element DNA, initiated by the p53 DNA binding domains. The slow oligomerization of free p53, competing with spontaneous denaturation, has implications for the possible regulation of p53 by binding proteins and DNA that affect tetramerization kinetics as well as equilibria.mass spectrometry ͉ protein-protein interactions ͉ slow association ͉ tetramerization domain T he tumor suppressor p53 is a transcription factor that is inactivated by mutation in some 50% of human cancers (1, 2). p53 consists of an unstructured N-terminal domain (residues 1-94) which points away from the rest of the protein (3), a folded core domain (residues 94-292), a linker to the tetramerization domain (residues 325-355) and an unstructured C-terminal domain . Nearly all of the mutations reside in its DNA-binding domain. Mutations can be either 'contact,' which remove residues that interact with DNA and lower affinity, or 'structural,' which destabilize the protein, sometimes with concomitant conformational changes (4). The transcriptionally active form of p53 is a homo-tetramer, linked via its tetramerization domains. Since most cancer mutations are located outside the tetramerization domain, in the core domain, the mutant (mut) proteins retain wild-type (wt) ability to form tetramers. Consequently the formation of hetero-tetramer complexes between wt and mut p53 is observed both in vivo and in vitro (5). It is thought that formation of hybrids between the wt and the mut protein is the cause of the trans-dominant effect of the mut over the wt in heterozygous cells (dominant-negative effect), either following somatic mutation or from birth as in the case of Li-Fraumeni syndrome (5, 6). In such events, the chances of initiating cancer are increased (7), and the p53 het...

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Section: Methodsmentioning

confidence: 99%

Ultraslow oligomerization equilibria of p53 and its implications

Natan

Hirschberg

Morgner

et al. 2009

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

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“…Mass Spectrometry-All samples were analyzed by nanoflow electrospray using capillaries prepared as described previously (23). The high viscosity of the ribosomal samples necessitated the use of needles with large tip orifices and a backing pressure of 10 -20 pounds/square inch.…”

Section: Methodsmentioning

confidence: 99%

Dissociation of Intact Escherichia coli Ribosomes in a Mass Spectrometer

Hanson¹,

Fucini²,

Ilag³

et al. 2003

Journal of Biological Chemistry

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We used mass spectrometry to identify proteins that are released in the gas phase from Escherichia coli ribosomes in response to a range of different solution conditions and cofactor binding. From solution at neutral pH the spectra are dominated by just 4 of the 54 ribosomal proteins (L7/L12, L11, and L10). Lowering the pH of the solution leads to the gas phase dissociation of four additional proteins as well as the 5 S RNA. Replacement of Mg 2؉ by Li ؉ ions in solutions of ribosomes induced the dissociation of 17 ribosomal proteins. Correlation of these results with available structural information for ribosomes revealed that a relatively high interaction surface area of the protein with RNA was the major force in preventing dissociation. By using the proteins that dissociate to probe their interactions with RNA, we examined different complexes of the ribosome formed with the elongation factor G and inhibited by fusidic acid or thiostrepton. Mass spectra recorded for the fusidic acid-inhibited complex reveal subtle changes in peak intensity of the proteins that dissociate. By contrast gas phase dissociation from the thiostrepton-inhibited complex is markedly different and demonstrates the presence of L5 and L18, two proteins that interact exclusively with the 5 S RNA. These results allow us to propose that the ribosome elongation factor-G complex inhibited by thiostrepton, but not fusidic acid, involves destabilization of 5 S RNA-protein interactions.

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“…It is often reported that the measured mass of large folded protein assemblies is somewhat higher then expected from the primary sequence. It has been speculated that the higher observed mass is because of trapping of small molecules or counterions in proteins (Nettleton et al, 1998;Rostom et al, 2000;Pinkse et al, 2003). This may especially be pronounced in multi-protein assemblies, which generally have cavities or holes.…”

Section: Electrospray Ionization Of Biomacromoleculesmentioning

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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Protein subunit interactions and structural integrity of amyloidogenic transthyretins: evidence from electrospray mass spectrometry

Cited by 162 publications

References 30 publications

Ultraslow oligomerization equilibria of p53 and its implications

Ultraslow oligomerization equilibria of p53 and its implications

Dissociation of Intact Escherichia coli Ribosomes in a Mass Spectrometer

Investigation of intact protein complexes by mass spectrometry

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