PELDOR Spectroscopy Distance Fingerprinting of the Octameric Outer‐Membrane Protein Wza from <i>Escherichia coli</i>.

Hagelueken, Gregor; Ingledew, W. John; Huang, Hexian; Petrovic-Stojanovska, Biljana; Whitfield, Chris; Mkami, Hassane El; Schiemann, Olav; Naismith, James H.

doi:10.1002/anie.200805758

Cited by 72 publications

(62 citation statements)

References 13 publications

(20 reference statements)

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“…The experimental and expected distributions for VcSiaP Q54R1/Q110R1 agree very well for both experiments (with and without Neu5Ac). A possible explanation for the shoulder at 30 Å is a second conformation of the R1 spin label, which has been frequently observed in available crystal structures of the R1 side chain (38)(39)(40).…”

Section: Building a Model Of Substrate-bound Vcsiapmentioning

confidence: 78%

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

Glaenzer

Peter

Thomas

et al. 2017

Biophysical Journal

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The tripartite ATP-independent periplasmic (TRAP) transporters are a widespread class of membrane transporters in bacteria and archaea. Typical substrates for TRAP transporters are organic acids including the sialic acid N-acetylneuraminic acid. The substrate binding proteins (SBP) of TRAP transporters are the best studied component and are responsible for initial high-affinity substrate binding. To better understand the dynamics of the ligand binding process, pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy was applied to study the conformational changes in the N-acetylneuraminic acid-specific SBP VcSiaP. The protein is the SBP of VcSiaPQM, a sialic acid TRAP transporter from Vibrio cholerae. Spin-labeled double-cysteine mutants of VcSiaP were analyzed in the substrate-bound and -free state and the measured distances were compared to available crystal structures. The data were compatible with two clear states only, which are consistent with the open and closed forms seen in TRAP SBP crystal structures. Substrate titration experiments demonstrated the transition of the population from one state to the other with no other observed forms. Mutants of key residues involved in ligand binding and/or proposed to be involved in domain closure were produced and the corresponding PELDOR experiments reveal important insights into the open-closed transition. The results are in excellent agreement with previous in vivo sialylation experiments. The structure of the spin-labeled Q54R1/L173R1 R125A mutant was solved at 2.1 Å resolution, revealing no significant changes in the protein structure. Thus, the loss of domain closure appears to be solely due to loss of binding. In conclusion, these data are consistent with TRAP SBPs undergoing a simple two-state transition from an openunliganded to closed-liganded state during the transport cycle.

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Section: Building a Model Of Substrate-bound Vcsiapmentioning

confidence: 78%

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

Glaenzer

Peter

Thomas

et al. 2017

Biophysical Journal

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“…Samples were diluted to 50% by the addition of ethylene glycol as cryoprotectant. Aliquots of 120 μl were transferred into quartz EPR tubes and quickly frozen as described earlier (14).…”

Section: Methodsmentioning

confidence: 99%

“…Thus, we have sought to apply new strategies for understanding MscS structure and function. Pulsed electron-electron double resonance (PEL-DOR; also called DEER) (13) is established for soluble proteins and has been applied to many proteins from monomers to octamers (14,15), to solvent exposed termini of transmembrane helices or loops that are embedded in lipid bilayers (16)(17)(18)(19), to transmembrane helices (20,21), and to 15 amino acid hydrophobic peptides in various lipid environments (22). Here we report the application of PELDOR to measure the separation of transmembrane structural elements in MscS in an attempt to experimentally resolve current disputes and thereby demonstrate the wider utility of PELDOR.…”

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confidence: 99%

Conformational state of the MscS mechanosensitive channel in solution revealed by pulsed electron–electron double resonance (PELDOR) spectroscopy

Pliotas

Ward

Branigan

et al. 2012

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

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The heptameric mechanosensitive channel of small conductance (MscS) provides a critical function in Escherichia coli where it opens in response to increased bilayer tension. Three approaches have defined different closed and open structures of the channel, resulting in mutually incompatible models of gating. We have attached spin labels to cysteine mutants on key secondary structural elements specifically chosen to discriminate between the competing models. The resulting pulsed electron-electron double resonance (PELDOR) spectra matched predicted distance distributions for the open crystal structure of MscS. The fit for the predictions by structural models of MscS derived by other techniques was not convincing. The assignment of MscS as open in detergent by PELDOR was unexpected but is supported by two crystal structures of spinlabeled MscS. PELDOR is therefore shown to be a powerful experimental tool to interrogate the conformation of transmembrane regions of integral membrane proteins.DEER | electron paramagenetic resonance | ion channels | dipolar coupling

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“…This excluded structures PDB:2W8H and PDB:2OU9 which do not display electron density because of a mismatch of more than 5% between predicted and reported R-values. In the former case, this may be due to crystal twinning, which was considered in refinement of the crystal structure [117]. Structure PDB:2IFX was excluded because of insufficient resolution (3.56 Å).…”

Section: Conformations Observed In Crystal Structuresmentioning

confidence: 99%

Conformational dynamics and distribution of nitroxide spin labels

Jeschke

2013

Progress in Nuclear Magnetic Resonance Spectroscopy

136

115

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Long-range distance measurements based on paramagnetic relaxation enhancement (PRE) in NMR, quantification of surface water dynamics near biomacromolecules by Overhauser dynamic nuclear polarization (DNP) and sensitivity enhancement by solid-state DNP all depend on introducing paramagnetic species into an otherwise diamagnetic NMR sample. The species can be introduced by site-directed spin labeling, which offers precise control for positioning the label in the sequence of a biopolymer. However, internal flexibility of the spin label gives rise to dynamic processes that potentially influence PRE and DNP behavior and leads to a spatial distribution of the electron spin even in solid samples. Internal dynamics of spin labels and their static conformational distributions have been studied mainly by electron paramagnetic resonance spectroscopy and molecular dynamics simulations, with a large body of results for the most widely applied methanethiosulfonate spin label MTSL. These results are critically discussed in a unifying picture based on rotameric states of the group that carries the spin label. Deficiencies in our current understanding of dynamics and conformations of spin labeled groups and of their influence on NMR observables are highlighted and directions for further research suggested.

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PELDOR Spectroscopy Distance Fingerprinting of the Octameric Outer‐Membrane Protein Wza from Escherichia coli.

Cited by 72 publications

References 13 publications

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

Conformational state of the MscS mechanosensitive channel in solution revealed by pulsed electron–electron double resonance (PELDOR) spectroscopy

Conformational dynamics and distribution of nitroxide spin labels

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