Real-Time Monitoring of Protein Complexes Reveals their Quaternary Organization and Dynamics

Painter, Alexander J.; Jaya, Nomalie; Basha, Eman; Vierling, Elizabeth; Robinson, Carol V.; Benesch, Justin L. P.

doi:10.1016/j.chembiol.2008.01.009

Cited by 72 publications

(78 citation statements)

References 43 publications

(57 reference statements)

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“…Insights into the assembly formation pathway may also be gained by disassembling intact structures into their building blocks followed by detailed analyses of the generated subcomplexes [65]. Recently, to track the assembly pathway of two dodecameric chaperone proteins, Painter et al introduced an automated approach for rapid and repeated monitoring of the reaction in real time [66]. The time resolution of this method is 32 s, far better than that achieved using offline methods, in which the dead time (i.e., the time between mixing of components and detection) is ϳ1 min.…”

Section: Assembly Dynamicsmentioning

confidence: 99%

How far can we go with structural mass spectrometry of protein complexes?

Sharon

2010

J. Am. Soc. Mass Spectrom.

100

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Physical interactions between proteins and the formation of stable complexes form the basis of most biological functions. Therefore, a critical step toward understanding the integrated workings of the cell is to determine the structure of protein complexes, and reveal how their structural organization dictates function. Studying the three-dimensional organization of protein assemblies, however, represents a major challenge for structural biologists, due to the large size of the complexes, their heterogeneous composition, their flexibility, and their asymmetric structure. In the last decade, mass spectrometry has proven to be a valuable tool for analyzing such noncovalent complexes. Here, I illustrate the breadth of structural information that can be obtained from this approach, and the steps taken to elucidate the stoichiometry, topology, packing, dynamics, and shape of protein complexes. In addition, I illustrate the challenges that lie ahead, and the future directions toward which the field might be heading. (J Am Soc Mass Spectrom 2010, 21, 487-500)

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Section: Assembly Dynamicsmentioning

confidence: 99%

How far can we go with structural mass spectrometry of protein complexes?

Sharon

2010

J. Am. Soc. Mass Spectrom.

100

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“…Additionally, the rapid analysis possible with ESI-MS makes it convenient for following subunit exchange in real time [38]. Despite these benefits, only a small number of studies applying ESI-MS to multimeric protein subunit exchange have been reported [39][40][41][42][43][44][45][46][47][48].…”

Section: Introductionmentioning

confidence: 99%

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

Mason

Jergic

et al. 2013

J. Am. Soc. Mass Spectrom.

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Escherichia coli single-stranded DNA-binding protein: nanoESI-MS studies of salt-modulated subunit exchange and DNA binding transactions AbstractSingle-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNA-binding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT)70, one molecule of (dT)35, or two molecules of (dT)35] was found to prevent SSB subunit exchange. Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry. Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNAbinding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT) 70 , one molecule of (dT) 35 or two molecules of (dT) 35 ] was found to prevent SSB subunit exchange.Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry.

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“…The dynamic nature of sHsp oligomers was long since established by monitoring the subunit exchange of sHsp heterooligomers by FRET (Bova et al 1997; and more recently by native MS (Painter et al 2008), and the dynamics and polydispersity of sHsps are clearly linked to their function of binding substrate proteins and protecting them from aggregation. Combining NMR, ion-mobility native mass spectrometry, and EM, structural models of the most abundant oligomeric states (24-, 26-, and 28-mers) and a model for the conversion between them have been proposed, providing a deeper understanding of the polydispersity and dynamics of sHsp structures (Baldwin et al 2011b).…”

Section: Introductionmentioning

confidence: 99%

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

Lambert

Rutsdottir

Hussein

et al. 2013

Cell Stress and Chaperones

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Small heat-shock protein chaperones are important players in the protein quality control system of the cell, because they can immediately respond to partially unfolded proteins, thereby protecting the cell from harmful aggregates. The small heat-shock proteins can form large polydisperse oligomers that are exceptionally dynamic, which is implicated in their function of protecting substrate proteins from aggregation. Yet the mechanism of substrate recognition remains poorly understood, and little is known about what parts of the small heat-shock proteins interact with substrates and what parts of a partially unfolded substrate protein interact with the small heat-shock proteins. The transient nature of the interactions that prevent substrate aggregation rationalize probing this interaction by crosslinking mass spectrometry. Here, we used a workflow with lysine-specific crosslinking and offline nano-liquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry to explore the interaction between the plant small heat-shock protein Hsp21 and a thermosensitive model substrate protein, malate dehydrogenase. The identified crosslinks point at an interaction between the disordered N-terminal region of Hsp21 and the C-terminal presumably unfolding part of the substrate protein.

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Real-Time Monitoring of Protein Complexes Reveals their Quaternary Organization and Dynamics

Cited by 72 publications

References 43 publications

How far can we go with structural mass spectrometry of protein complexes?

How far can we go with structural mass spectrometry of protein complexes?

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

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