Structure and function of the membrane protein Opa60 by solid-state NMR
by Marcel Christian Forster
Date of Examination:2021-07-06
Date of issue:2021-10-18
Advisor:Dr. Loren Andreas
Referee:Prof. Dr. Christian Griesinger
Referee:Prof. Dr. Ralf Ficner
Referee:Prof. Dr. Markus Zweckstetter
Referee:Prof. Dr. Helmut Grubmüller
Referee:Prof. Dr. Marina Bennati
Persistent Address:
http://dx.doi.org/10.53846/goediss-8862
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Abstract
English
Studying the structure of transmembrane (TM) proteins is an important task since many of them are drug targets and are involved in essential cellular functions. However, new techniques are urgently needed, as TM proteins are not amenable to conventional techniques. The recent development of ultrafast magic angle spinning solid-state NMR with proton detection provides a promising avenue. The protein Opa60 is a β-barrel TM protein found in the outer membrane of the human pathogen Neisseria gonorrhoeae and mediates adhesion to human host cells via the hCEACAM-family of TM proteins with its large extracellular loops. Its structure has been determined previously in detergent micelles by solution NMR. However, structural insight in a native-like environment is needed to study the binding of Opa60 to its receptor under more physiological conditions and therefore, proton-detected solid-state NMR was chosen in this thesis as a technique to study Opa60 in DMPC lipid bilayers. In this work, the structure of the β-barrel was successfully determined and shows an extension of the length of the β-strands compared to the solution structure. The loops retain their dynamic behavior and are not visible in cross polarization based solid-state NMR experiments. The binding of Opa60 to the N-terminal domain of hCEACAM1 (hCEACAM1-N) could not be shown in lipid bilayers, however solution NMR data indicated a possible interaction in detergent. Opa60 in DMPC was compared with Opa60 in two different LPS species and Kdo2-lipid A, which resemble the native outer membrane environment of N. gonorrhoeae, but no major structural changes were apparent from the spectra. In the future, the assignments should be completed and likely reveal the extension of the complete β-barrel to match the thickness of the lipid bilayer. The receptor binding conditions will need to be investigated systematically. Alternatively, a solution NMR assignment of hCEACAM1-N can be conducted and used as a starting point to target the binding surface of Opa60 employing various isotopic labeling schemes. Method development in solid-state NMR is ongoing and of high importance. Techniques for accurate internuclear distance determination are sought for. In this thesis, a new technique was developed termed TRansferred Rotational DOuble Resonance (TREDOR), based on the TEDOR and REDOR sequences. In TREDOR, both starting and transferred signal are co-acquired and this enables a single parameter fit to the internuclear distance. After calibrating the fitting, the accuracy and ease of TREDOR was demonstrated on the microcrystalline protein SH3. The structure was determined using only TREDOR-derived distances and TALOS-N angle restraints. The superiority of TREDOR over TEDOR was shown in terms of distance accuracy when compared to a known crystal structure of SH3. Moreover, TREDOR can be applied with only a single mixing time, drastically reducing the necessary measurement time. TREDOR was applied to the TM protein Opa60, and one long-distance contact was found. The technique is mostly limited by strong peak overlap, as seen for Opa60. The application of TREDOR to other challenging systems will likely prove it to be a valuable tool in structural biology.
Keywords: Solid-State NMR; Transmembrane protein; Lipid bilayer; Opa60; Neisseria; TREDOR; CEACAM; TEDOR; REDOR