Development and Application of CP-ENDOR Spectroscopy on Protein Radicals
von Isabel Bejenke
Datum der mündl. Prüfung:2019-07-04
Betreuer:Prof. Dr. Marina Bennati
Gutachter:Prof. Dr. Marina Bennati
Gutachter:Prof. Dr. Christian Griesinger
EnglischElectron-nuclear double resonance (ENDOR) spectroscopy is the method of choice for detecting magnetic nuclei in biomolecules which contain an unpaired electron spin. However, due to its low gyromagnetic ratio, the detection of Deuterium couplings is a particular challenge for ENDOR spectroscopy. The standard ENDOR sequences suﬀer from either low sensitivity or line shape distortions. Yet, deuterium nuclei are among the most interesting targets for ENDOR spectroscopy. Hydrogen-bond environments of biomolecules can be investigated at the molecular scale by 2H ENDOR spectroscopy in combination with H2O to D2O buﬀer exchange. Thus, aiming at improved sensitivity and/or spectral resolution, alternatives to the conventional ENDOR sequences have been proposed. The cross-polarization edited ENDOR approach (CP-ENDOR) merges electron-nuclear cross polarization with ENDOR spectroscopy and generates an alternative polarization transfer scheme for it. In this thesis, the capability of the CP-ENDOR sequence to detect small hyperﬁne couplings between an electron spin and deuterium nuclei with high sensitivity is demonstrated at 94GHz/3.4T. The CP-ENDOR polarization transfer mechanism involving deuterium nuclei was established from single crystal studies of a deuterated malonic acid radical. The matching conditions for cross polarization and the CP-ENDOR intensities were determined experimentally and validated from analytical and numerical predictions. The sensitivity for detecting small hyperﬁne couplings in CP-ENDOR is signiﬁcantly improved when omitting the initial pi/2 pulse of the sequence. The modiﬁed sequence has been named “without preparation pulse” (WOP) CP-ENDOR. Its performance was evaluated on the EPR standard organic radical 2H-BDPA in crystalline powder form and resulted in an improvement of the signal-to-noise ratio by a factor of approximately ﬁvein comparison to the standard CP-ENDOR. The enhancement is attributed to diﬀerent excitation proﬁles of the CP-ENDOR sequences, causing a central blind spot of diﬀerent width in the ENDOR spectra. The width of the central blind spot of WOP CP-ENDOR is determined from analytical and numerical calculations and scales with the strength of the applied microwave irradiation pulse during the CP-step of the sequence. Representative studies of the radical intermediate ND2Y731• in E.coli RNR illustrate the advantages of WOP CP-ENDOR in terms of signal-to-noise ratio and line shape in comparison to the well-established ENDOR sequences. Particularly, the WOP CPENDOR sequence has unmasked hyperﬁne tensor features of the amino group which are usually distorted by Mims blind spots in the ENDOR spectrum recorded with the wellestablished Mims ENDOR sequence. These advantages have allowed the establishment of the planarity of the functional group.
Keywords: EPR; ENDOR; NMR; hyperfine spectroscopy; cross polarization; spin polarization; RNR