Spin labeling EPR

Klare, Johann P.; Steinhoff, Heinz‐Jürgen

doi:10.1007/s11120-009-9490-7

Cited by 222 publications

(175 citation statements)

References 114 publications

Supporting

Mentioning

174

Contrasting

Unclassified

Order By: Relevance

“…Here, pulsed electron-electron double resonance (PELDOR) spectroscopy (also known as double electron-electron resonance (DEER) spectroscopy) was applied to analyze the structure of the P domain of VcSiaPQM, a Neu5Ac transporter from Vibrio cholerae, in solution (13,14). Site-directed spin labeling (15,16) was used to introduce nitroxide spin labels at positions that allow to readily distinguish the open-and closed states of the protein. These labeled forms were then used to study the structure of the protein in solution.…”

Section: Introductionmentioning

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

View full text Add to dashboard Cite

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.

show abstract

Section: Introductionmentioning

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

View full text Add to dashboard Cite

show abstract

“…They ruled out oxidative radical coupling with 83 constituents of SOM as an alternative reaction pathway. 84 ESR spin labeling with stable nitroxide spin labels has successfully been applied since many 85 years for investigating the structure and dynamics of biological macromolecules and assemblies, 86 particularly membranes and proteins [24,25]. Stable nitroxide radicals can also be used for 87 studying the interaction of xenobiotics in soil and sediment systems.…”

Section: Abstract: 31mentioning

confidence: 99%

Kinetics of Rapid Covalent Bond Formation of Aniline with Humic Acid: ESR Investigations with Nitroxide Spin Labels

et al. 2016

Self Cite

View full text Add to dashboard Cite

11The bioavailability of many soil contaminants depends on their interaction with the soil organic 12 matter. The paper presents a new approach of using stable paramagnetic spin labels for 13 investigating the kinetics of covalent binding of specific xenobiotic functional groups with humic 14 acids, a major organic matter fraction. Leonardite humic acid (LHA) was incubated with the 15 nitroxide spin labels amino-TEMPO (4-amino-2,2,6,6-Tetramethylpiperidin-1-oxyl) and anilino-16 NO (2,5,5-Trimethyl-2-(3-aminophenyl)pyrrolidin-1-oxyl), respectively, which contain an 17 aliphatic or aromatic functionality susceptible to interaction with LHA. Electron spin resonance 18 2 (ESR) spectra of LHA samples without and with the enzyme laccase were recorded at X-band 19 frequency (9.43 GHz) at room temperature and neutral pH. Binding was detected by a 20 pronounced broadening of the spectral lines after incubation of LHA for both spin labels. The 21 development of a broad signal component in the spectrum of anilino-NO indicated the 22 immobilization due to strong binding of the aniline group. The reorientational correlation time of 23 bound anilino-NO is more than two orders of magnitude greater than that of the free label. The 24 ratio of the amount of bound to the unbound species was used to determine the kinetics of the 25 covalent bond formation. Reaction rate constants of 0.16 min -1 and 0.01 min -1 were determined 26 corresponding to half-times of 4.3 min and 69.3 min, respectively. Treatment of LHA with 27 laccase enhanced the amount of the reacting anilino-NO species by a factor of 7.6, but left the 28 reaction rate unaltered. Oxidative radical coupling was excluded by using the spin trap agent n-29 tert-butyl-alpha-phenylnitrone. 30

show abstract

“…14, [24][25][26][27] The commonly used nitroxides 28 as spin labels 29,30 are also smaller than fluorophores, and thus interfere less with the system of interest. 31,32 On a molecular basis, p-conjugated oligomers are referred to as rigid rods, 33 which makes them desirable as building blocks for (supra-)molecular architectures.…”

Section: Introductionmentioning

confidence: 99%

Probing of chain conformations in conjugated polymer nanoparticles by electron spin resonance spectroscopy

Hintze

Schütze

Drescher

et al. 2015

Phys. Chem. Chem. Phys.

View full text Add to dashboard Cite

show abstract

Spin labeling EPR

Cited by 222 publications

References 114 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

Kinetics of Rapid Covalent Bond Formation of Aniline with Humic Acid: ESR Investigations with Nitroxide Spin Labels

Probing of chain conformations in conjugated polymer nanoparticles by electron spin resonance spectroscopy

Contact Info

Product

Resources

About