Recent Advances in X-Ray Hydroxyl Radical Footprinting at the Advanced Light Source Synchrotron

Morton, Simon A.; Gupta, Sayan; Kim, Young-Mo; Ralston, Corie Y.

doi:10.2174/0929866526666181128125725

Cited by 5 publications

(4 citation statements)

References 25 publications

(27 reference statements)

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“…The analysis yielded quantitative measures for conformational selection and conserved residue-reaction pathways. As an adjunct to MD analysis, we used time-resolved X-ray footprinting (XFMS) (35,36) to examine solution exposure of selected positions in FliMN-CheY fusion proteins, mimics of the FliMN-captured state of CheY and shown previously to bind to FliN (37). The use of fusion constructs simplified interpretation of the XFMS data by preventing solution heterogeneity that might otherwise dominate mixtures of weak-binding components.…”

Section: Introductionmentioning

confidence: 99%

Allosteric priming ofE. coliCheY by the flagellar motor protein FliM

Wheatley

Pandini

Gupta

et al. 2019

Preprint

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Phosphorylation of Escherichia coli CheY couple's chemoreceptor output to flagellar motor response. The N-termini of FliM subunits (FliMN) in the flagellar rotor prime CheY to bind FliN thereby inducing cooperative switching, an essential feature of bacterial chemotaxis. We analyzed molecular dynamics {MD} trajectories to identify networks of residues involved in the long-range allosteric activation of CheY by FliMN. The CheY backbone was partitioned into four dynamically coordinated sectors, with activation tracked by changes in sector size and interactions. Bound FliMN closed the central α4-β4 loop hinge to strengthen correlations between sectors around its binding interface and D57 phosphorylation site. Inward W58 sidechain movements adjacent to the CheY D57 phosphorylation site were coupled to corresponding K91 and Y106 sidechain movements at the FliMN interface. Studies of the constitutively active CheY D13K-Y106W double mutant have related its structural changes with invivo signaling properties. The MD revealed that D13K-Y106W fused the phosphorylation site and FliMN binding sectors into a new surface-exposed sector and locked the 106W sidechain in the innermost rotamer configuration in CheY-FliMN complexes. X-ray foot-printing with mass spectroscopy exploited FliMN-CheY fusion proteins to validate the concerted sidechain internalization of W58, K91 and Y106 triggered by bound FliMN and increased by D13K-Y106W. Oxidation rate was correlated with the solvent accessible surface area, with K109, another central element of the allosteric relay, an outlier likely due to hydrogen bonding. The measurements indicated the fusion proteins were an effective mimic of the crystallized complexes used for the MD simulations. In absence of the D13K-Y106W mutations, CheY Y106 sampled multiple inward rotamer states, but their coupling to backbone dynamics required bound FliMN to prolongation inward state lifetimes by bound FliMN. Thus, as simulations have found for kinases, control of CheY activation by aromatic residue reorientation is more subtle than a binary ON-OFF switch. Statement of SignificanceThe chemotaxis phospho-protein CheY is activated at the flagellar motor by the Nterminus (FliMN) of FliM subunits. Crystal structures of FliMN.CheY complexes with and without the phosphomimic D13K-Y106W double-mutation both have the residue 106 sidechain in the same IN orientation at the FliMN interface. Additional factors that explain activation were identified by atomistic simulations based on the crystal structures. Free CheY samples both IN and OUT Y106 rotamer states, but bound FliMN increases multiple IN-state lifetimes to alter backbone dynamics. D13K-Y106W triggers further alterations to select the W106 state seen in the crystals and create novel binding surfaces. Observed changes in sidechain position along the allosteric relay reported by the crystals were predicted and validated by X-ray foot-printing with mass-spectroscopy.

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Section: Introductionmentioning

confidence: 99%

Allosteric priming ofE. coliCheY by the flagellar motor protein FliM

Wheatley

Pandini

Gupta

et al. 2019

Preprint

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“…The set-up will later be used for more challenging systems, such as in vivo cell studies, large complexes, and membrane proteins, as well as micro-second time resolved in situ hydroxyl labeling studies. Currently, the development of a micro-focus capability at the ALS at beamline 3.3.1 is in progress 39 . This beamline will host the only dedicated XFMS program on the West Coast.…”

Section: Discussionmentioning

confidence: 99%

“…Currently, the development of a microfocus capability at the ALS at beamline 3.3.1 is in progress. 39 This beamline will host the only dedicated XFMS program on the West Coast. Beamline 3.3.1 is located on a white light bend magnet source with an acceptance of 6.2 × 2 mrad and a planned Pt-coated toroidal focusing mirror centered at 11.1 m (2/3 of the distance) from the source.…”

Section: ■ Conclusion and Future Directionsmentioning

confidence: 99%

Development of Container Free Sample Exposure for Synchrotron X-ray Footprinting

et al. 2019

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The method of X-ray footprinting and mass spectrometry (XFMS) on large protein assemblies and membrane protein samples requires high flux density to overcome the hydroxyl radical scavenging reactions produced by the buffer constituents and the total protein content. Previously, we successfully developed microsecond XFMS using microfluidic capillary flow and a microfocused broadband X-ray source at the Advanced Light Source synchrotron beamlines, but the excessive radiation damage incurred when using capillaries prevented the full usage of a high-flux density beam. Here we present another significant advance for the XFMS method: the instrumentation of a liquid injection jet to deliver container free samples to the X-ray beam. Our preliminary experiments with a liquid jet at a bending magnet X-ray beamline demonstrate the feasibility of the approach and show a significant improvement in the effective dose for both the Alexa fluorescence assay and protein samples compared to conventional capillary flow methods. The combination of precisely controlled high dose delivery, shorter exposure times, and elimination of radiation damage due to capillary effects significantly increases the signal quality of the hydroxyl radical modification products and the dose–response data. This new approach is the first application of container free sample handling for XFMS and opens up the method for even further advances, such as high-quality microsecond time-resolved XFMS studies.

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“…There are currently two major footprinting centers in the United States: the National Synchrotron Light Source (NSLS) and the Advanced Light Source (ALS). The detailed development of XFMS was summarized in several recent reviews in 2019 and 2020 [49,50,63]. In this section, we outline the latest devel-opments and future promise of XFMS to study MP structure and dynamics.…”

Section: Synchrotron Hrfmentioning

confidence: 99%

Advances in mass spectrometry‐based footprinting of membrane proteins

Gross

2022

Proteomics

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Structural biology is entering an exciting time where many new high‐resolution structures of large complexes and membrane proteins (MPs) are determined regularly. These advances have been driven by over 15 years of technological improvements, first in macromolecular crystallography, and recently in cryo‐electron microscopy. Obtaining information about MP higher order structure and interactions is also a frontier, important but challenging owing to their unique properties and the need to choose suitable detergents/lipids for their study. The development of mass spectrometry (MS), both instruments and methodology in the past 10 years, has also advanced it as a complementary method to study MP structure and interactions. In this review, we discuss advances in MS‐based footprinting for MPs and highlight recent methodologies that offer new promise for MP study by chemical footprinting and mass spectrometry.

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Recent Advances in X-Ray Hydroxyl Radical Footprinting at the Advanced Light Source Synchrotron

Cited by 5 publications

References 25 publications

Allosteric priming ofE. coliCheY by the flagellar motor protein FliM

Allosteric priming ofE. coliCheY by the flagellar motor protein FliM

Development of Container Free Sample Exposure for Synchrotron X-ray Footprinting

Advances in mass spectrometry‐based footprinting of membrane proteins

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