Trityl Radicals: Spin Labels for Nanometer‐Distance Measurements

Reginsson, Gunnar W.; Kunjir, Nitin C.; Sigurdsson, Snorri Th.; Schiemann, Olav

doi:10.1002/chem.201203014

Cited by 123 publications

(151 citation statements)

References 35 publications

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“…Therefore, if a K d can be determined, it might differ from a K d that was determined at room temperature, depending on the k on and k off rates compared to the time needed for the freezing process. The recently developed trityl spin labels, which can be used at room temperature might be a possibility to avoid this problem (42)(43)(44). However, these labels are considerably larger than the MTSSL label and are currently not used on a routine basis.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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 use of conventional nitroxide radicals as spin labels in most cases restricts the information extracted from a single sample to one distance distribution measured between two labeled sites. Combining different types of spin labels can enhance the information content accessible per one spin-labeled biomolecular sample [18][19][20][21][22][23][24][25][26]. The combination of nitroxide radicals with lanthanide ions, which provides good sensitivity, excellent spectroscopic selection and robust performance, appears to be particularly advantageous [22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Distance determination from dysprosium induced relaxation enhancement: a case study on membrane-inserted WALP23 polypeptides

et al. 2013

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Abstract. Membrane incorporated synthetic α-helical polypeptides labeled with Dy(III) chelate complexes and nitroxide radicals were studied by the inversion recovery (IR) technique and Dy(III)-nitroxide distances were obtained. A comparison of obtained distances with the previously reported Gd(III)-nitroxide pulse double electron-electron resonance (DEER or PELDOR) calibration data was performed and revealed reliability of the IR-based technique for the distance determination in membrane-incorporated biomacromolecules. The presented distance determination technique is 'spectroscopically orthogonal' to DEER-based distance measurements and can be potentially combined with DEER to study multiply spin-labeled biomacromolecules. The key steps of the data processing, the types of obtained distance information and the areas of possible application of the technique are discussed.

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“…For the first time applicability of trityl radicals as spin labels for nanometer-distance measurements at cryogenic temperatures has been exemplified by poly(para-phenyleneethynylene)s polymers ( Fig. 9; Reginsson et al, 2012). The trityl-nitroxide biradical and Figure 9 Structures of trityl-trityl and trityl-nitroxide biradicals.…”

Section: Spin Labels For Room-temperature Epr Distance Measurementsmentioning

confidence: 98%

Development and Application of Spin Traps, Spin Probes, and Spin Labels

Bagryanskaya

Krumkacheva

Fedin

et al. 2015

Methods in Enzymology

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Trityl Radicals: Spin Labels for Nanometer‐Distance Measurements

Cited by 123 publications

References 35 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

Distance determination from dysprosium induced relaxation enhancement: a case study on membrane-inserted WALP23 polypeptides

Development and Application of Spin Traps, Spin Probes, and Spin Labels

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