General Synthesis of Persistent Trityl Radicals for EPR Imaging of Biological Systems

Reddy, T. Jagadeeswar; Iwama, Tetsuo; Halpern, Howard J.; Rawal, Viresh H.

doi:10.1021/jo011068f

Cited by 177 publications

(224 citation statements)

References 15 publications

(10 reference statements)

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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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“…General synthesis of these types of products was also recently described. 164 The EPR spectrum of these compounds displays one single line with very small 13 C satellite lines. 156 One important interesting characteristic of triarylmethyl radicals is the narrowness of their EPR linewidth (typically less than 100 mGauss).…”

Section: Soluble Paramagnetic Materialsmentioning

confidence: 97%

Assessment of tumor oxygenation by electron paramagnetic resonance: principles and applications

2004

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This review paper attempts to provide an overview of the principles and techniques that are often termed electron paramagnetic resonance (EPR) oximetry. The paper discusses the potential of such methods and illustrates they have been successfully applied to measure oxygen tension, an essential parameter of the tumor microenvironment. To help the reader understand the motivation for carrying out these measurements, the importance of tumor hypoxia is first discussed: the basic issues of why a tumor is hypoxic, why these hypoxic microenvironments promote processes driving malignant progression and why hypoxia dramatically influences the response of tumors to cytotoxic treatments will be explained. The different methods that have been used to estimate the oxygenation in tumors will be reviewed. To introduce the basics of EPR oximetry, the specificity of in vivo EPR will be discussed by comparing this technique with NMR and MRI. The different types of paramagnetic oxygen sensors will be presented, as well as the methods for recording the information (EPR spectroscopy, EPR imaging, dynamic nuclear polarization). Several applications of EPR for characterizing tumor oxygenation will be illustrated, with a special emphasis on pharmacological interventions that modulate the tumor microenvironment. Finally, the challenges for transposing the method into the clinic will also be discussed.

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“…In particular, the lineshape is characteristic of an OE in which the sign of the DNP enhancement does not depend upon the offset of the microwave frequency. Interestingly, trityl OX063 radical, 31 which was designed to optimize Overhauser effects in solution by eliminating hyperfine couplings, 32,33 shows only SE enhancements at ω 0S ± ω 0I . Thus, the OE effect appears to require the presence of multiple hyperfine or dipolar couplings that permit zero quantum (ZQ) or double quantum (DQ) relaxation, respectively, and Overhauser enhancements.…”

Section: Introductionmentioning

confidence: 99%

Overhauser effects in insulating solids

Can

Caporini

Mentink-Vigier

et al. 2014

The Journal of Chemical Physics

172

226

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We report magic angle spinning, dynamic nuclear polarization (DNP) experiments at magnetic fields of 9.4 T, 14.1 T, and 18.8 T using the narrow line polarizing agents 1,3-bisdiphenylene-2-phenylallyl (BDPA) dispersed in polystyrene, and sulfonated-BDPA (SA-BDPA) and trityl OX063 in glassy glycerol/water matrices. The 1 H DNP enhancement field profiles of the BDPA radicals exhibit a significant DNP Overhauser effect (OE) as well as a solid effect (SE) despite the fact that these samples are insulating solids. In contrast, trityl exhibits only a SE enhancement. Data suggest that the appearance of the OE is due to rather strong electron-nuclear hyperfine couplings present in BDPA and SA-BDPA, which are absent in trityl and perdeuterated BDPA (d 21 -BDPA). In addition, and in contrast to other DNP mechanisms such as the solid effect or cross effect, the experimental data suggest that the OE in non-conducting solids scales favorably with magnetic field, increasing in magnitude in going from 5 T, to 9.4 T, to 14.1 T, and to 18.8 T. Simulations using a model two spin system consisting of an electron hyperfine coupled to a 1 H reproduce the essential features of the field profiles and indicate that the OE in these samples originates from the zero and double quantum cross relaxation induced by fluctuating hyperfine interactions between the intramolecular delocalized unpaired electrons and their neighboring nuclei, and that the size of these hyperfine couplings is crucial to the magnitude of the enhancements. Microwave power dependent studies show that the OE saturates at considerably lower power levels than the solid effect in the same samples. Our results provide new insights into the mechanism of the Overhauser effect, and also provide a new approach to perform DNP experiments in chemical, biophysical, and physical systems at high magnetic fields. INTRODUCTIONThe last decade has witnessed a renaissance in the use of high frequency dynamic nuclear polarization (DNP) to enhance sensitivity in nuclear magnetic resonance (NMR) experiments. In particular, the development of gyrotron and other high frequency microwave sources permits DNP to be performed at magnetic fields used in contemporary NMR experiments (5-20 T). 1-8 To date these experiments, which have focused mostly on insulating solids formed from glassy, frozen solutions of proteins and other nonconducting materials, have relied primarily on narrow line monoradicals and the solid effect (SE) 1, 9-11 or nitroxide biradicals and the cross effect (CE) [12][13][14][15][16][17][18] to mediate the polarization process. These approaches have resulted in large signal enhancements and have enabled many experiments that would otherwise be impossible. [19][20][21][22][23] by Overhauser 25 and confirmed by Carver and Slichter, 26 has not been identified or utilized during the course of this renaissance. Although the possibility of an OE in insulator was discussed by Abragam, 27 the conventional wisdom is that Overhauser DNP is important only in systems with mobile electrons ...

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General Synthesis of Persistent Trityl Radicals for EPR Imaging of Biological Systems

Cited by 177 publications

References 15 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

Assessment of tumor oxygenation by electron paramagnetic resonance: principles and applications

Overhauser effects in insulating solids

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