Research Group Reif

Projects AG Reif

1) Structural Characterization of beta-amyloid peptides (Abeta)

Alzheimer's disease is the most common form of age-related neurodegenerative disorder. Abeta is obtained after processing of APP, the amyloid precursor protein. We study different fragments of Abeta using solid-state NMR methods in order to better understand the mechanisms which lead to the formation of these aggregates. For this purpose, we compare the structure of different Abeta fragments.

Using chemical shift perturbation and paramagnetic relaxation enhancement, we determine the binding site of small molecules that interact with Abeta amyloid in different aggregation states. The results obtained from these experiments will allow to design more potent binders which facilitate the diagnosis of the disease. 

         

2) Membrane Proteins

Resistance to toxicants is mediated by membrane proteins that can translocate substrates across cell membranes. Therefore, these transporters play an important role in resistance to antitumor chemotherapeutic agents or resistance to antibiotics. We focus on studying secondary transporters which transport toxicants versus a proton gradient. In contrast, primary transporters, like ABC transporters, no ATP is required for activity. We are especially interested in the class of small multidrug resistance (SMR) membrane proteins which contain only four transmembrane helices. The goal of this project is to obtain a 3D structure of this membrane protein using solid-state NMR spectroscopy. This way, we hope to obtain a mechanistic understanding of the function of these membrane proteins. 

The E.Coli multidrug resistance transporter (EmrE) is a secondary transporter which transports polyaromatic compounds out of a cell against a proton gradient. Bottom: 13C,13C correlation spectra showing the threonine spectral region of a EmrE sample that was reconstituted in lipid membranes (Agarwal et al., BBA-Biomembranes 2007).

     

3) Large Protein Complexes

The relaxation rate of a resonance in solution-state NMR is determined by the tumbling rate of the molecule. Typically, line widts increase for larger molecules, making it increasingly difficult to characterize molecules which are larger than 100 kDa. Solid-state NMR is not limited by tumbling correlation time, as the molecules are immobilized. Therefore, the experimental line width is independent of the size of the molecule. Immobilization of the molecule can be achieved by crystallization, but also by just increasing the viscosity of the solution. This approach was demonstrated using of the small heat shock protein αB-crystallin. This techniques opens new perspectives for the investigation of large protein complexes, as ligands do not have to be co-precipitated to study protein-protein interactions. 

MAS NMR 13C,13C correlations carried out for the small heat shock protein αB-Crystallin (600 kDa) in solution (Mainz et al., J. Am. Chem. Soc. 2009)

        

4) Solid-State NMR Methods

So far, solid-state NMR experiments that measure distances or torsion angles between heteroatoms, are restricted to doubly labeled peptide or protein samples. We develop experiments that are suitable to characterize the structure of uniformly labeled peptides proteins. Furthermore, we develop labeling concepts that allow to detect protons with high sensitivity. These strategies rely on perdeuteration of the protein with backsubstitution of the exchangeable deuterons with protons from the solvent.

 One focus of the research is the quantification of dynamics. Solid-state NMR is like no other method suited to describe dynamics. In solution, most of the relaxation is due to overall tumbling. By contrast, relaxation in the solid-state is only due to local structural fluctuations. 

HSQC spectrum of a-spectrin SH3 domain obtained in the solid-state. The obtainable resolution is comparable to a the resolution that is achieved in solution-state NMR for an intermediate sized protein. (Chevelkov et al., Angewandte Chemie Int. Edt. 2006; Linser et al., J. Am. Chem. Soc. 2010)

 

Cross sections along the 15N dimension in a HSQC experiment that was recorded without scalar decoupling in t1. Accordingly, signals are split into doublets. Intensities of the 15N-1H(α) and 15N-1H(β) spin states are assymetric due to dipole-CSA cross correlated relaxation.

 

In collaboration with Prof. Nikolai Skrynnikov, Purdue University, we compare the dynamic properties of the α-spectrin SH3 domain in the crystalline state and in solution. We find that the motional properties are highly similar. This opens new perspectives for the dynamic characterization of a protein. 

Correlation of methyl 13C-R1 relaxation rates for the α-spectrin SH3 domain in solution and in the solid-state. Even without the correction term which takes into account overall tumbling, the relaxation rates are very similar (Agarwal et al., J. Am. Chem. Soc. 2008).