Einbau von Rezeptoren in Membran-Nanodisks

In dieser Arbeit wurde sensorisches Rhodopsin II (SRII) und der Photorezeptorkomplex, SRII mit seinem Transducer HtrII in Lipid Nanodisks eingelagert. Nanodisks bestehen aus einer Phospholipid Doppelschicht die von einem Proteinmantel umrandet wird und bilden eine wasserlösliche Modelmembran. Verschiedene biochemische Parameter wurden optimiert um einen möglichst effizienten Einbau der Membranproteine und anschließender Aufreinigung der Disks, zu erreichen. Der Einbau der relevanten Trimer von Dimer Stöchiometrie des SRII/HtrII Photorezeptorkomplex konnte deutlich im Elektronenmikroskop sichtbar gemacht werden. Außerdem wurde durch Messung der Kinetik des Photozyklus des SRII sowie des Komplexes in Nanodisks die Aktivität der Membranproteine nachgewiesen. Abstract: In this work, sensory rhodopsin II (SRII) and the photoreceptor complex, SRII with its transducer HtrII were incorporated into lipid nanodiscs. Nanodiscs consist of a phospholipid bilayer surrounded by a protein shell and form a water-soluble model membrane. Various biochemical parameters were optimized to achieve the most efficient membrane protein reconstitution and subsequent purification of the discs. The incorporation of the relevant trimers of dimers stoichiometry of the SRII/HtrII photoreceptor complex was clearly visualized by electron microscopy. Furthermore, the protein activity was determined by measurements of SRII photocycle in nanodiscs

Probing membrane protein insertion into lipid bilayers by solid-state NMR.

Determination of the environment surrounding a protein is often key to understanding function, and can also be used to infer structural properties of the protein itself. Using proton-detected solid-state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic-angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site-specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid-protein transfer rate of 6 s¯¹ was determined for residues protected from exchange. Transfer within the protein, as tracked in the ¹⁵N-¹H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s-1 for several resolved residues, explaining the site specificity

NMR spectroscopic assignment of backbone and side-chain protons in fully protonated proteins: Microcrystals, sedimented assemblies, and amyloid fibrils.

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR spectroscopy with 100–111 kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα–Hα detection block was developed and applied for the sequence-specific backbone and aliphatic side-chain resonance assignment using only 500 μg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration