Karsten Seidel

Characterization of Alzheimer's-like Paired Helical Filaments from the Core Domain of Tau Protein Using Solid-State NMR Spectroscopy

The polymerization of the microtubule-associated protein tau into paired helical filaments (PHFs) represents one of the hallmarks of Alzheimers disease. We employed solid-state nuclear magnetic resonance (NMR) to investigate the structure and dynamics of PHFs formed in vitro by the three-repeat-domain (K19) of protein tau, representing the core of Alzheimer PHFs. While N and C termini of tau monomers in PHFs are highly dynamic and solvent-exposed, the rigid segment consists of three major beta-strands. Combination of through-bond and through-space ssNMR transfer methods with water-edited ((15)N, (13)C) and ((13)C, (13)C) correlation experiments suggests the existence of a fibril core that is largely built by repeat unit R3, flanked by surface-exposed units R1 and R4. Solid-state NMR, circular dichroism, and the fibrillization behavior of a K19 mutant furthermore indicate that electrostatic interactions play a central role in stabilizing the K19 PHFs.

Structural rearrangements of membrane proteins probed by water-edited solid-state NMR spectroscopy

We show that water-edited solid-state NMR spectroscopy allows for probing global protein conformation and residue-specific solvent accessibility in a lipid bilayer environment. The transfer dynamics can be well described by a general time constant, irrespective of protein topology and lipid environment. This approach was used to follow structural changes in response to protein function in the chimeric potassium channel KcsA-Kv1.3. Data obtained as a function of pH link earlier biochemical data to changes in protein structure in a functional bilayer setting.

Protein refolding is required for assembly of the type three secretion needle

Pathogenic Gram-negative bacteria use a type three secretion system (TTSS) to deliver virulence factors into host cells. Although the order in which proteins incorporate into the growing TTSS is well described, the underlying assembly mechanisms are still unclear. Here we show that the TTSS needle protomer refolds spontaneously to extend the needle from the distal end. We developed a functional mutant of the needle protomer from Shigella flexneri and Salmonella typhimurium to study its assembly in vitro. We show that the protomer partially refolds from α-helix into β-strand conformation to form the TTSS needle. Reconstitution experiments show that needle growth does not require ATP. Thus, like the structurally related flagellar systems, the needle elongates by subunit polymerization at the distal end but requires protomer refolding. Our studies provide a starting point to understand the molecular assembly mechanisms and the structure of the TTSS at atomic level.

High‐Resolution Solid‐State NMR Studies on Uniformly [13C,15N]‐Labeled Ubiquitin

Understanding of the effects of intermolecular interactions, molecular dynamics, and sample preparation on high‐resolution magic‐angle spinning NMR data is currently limited. Using the example of a uniformly [13C,15N]‐labeled sample of ubiquitin, we discuss solid‐state NMR methods tailored to the construction of 3D molecular structure and study the influence of solid‐phase protein preparation on solid‐state NMR spectra. A comparative analysis of 13C′, 13Cα, and 13Cβ resonance frequencies suggests that 13C chemical‐shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. Our results can be refined by additional solid‐state NMR techniques and serve as a reference for ongoing efforts to characterize the structure and dynamics of (membrane) proteins, protein complexes, and other biomolecules by high‐resolution solid‐state NMR.

Structural characterization of Ca2+-ATPase-bound phospholamban in lipid bilayers by solid-state nuclear magnetic resonance (NMR) spectroscopy.

Phospholamban (PLN) regulates cardiac contractility by modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. While PLN and SERCA1a, an isoform from skeletal muscle, have been structurally characterized in great detail, direct information about the conformation of PLN in complex with SERCA has been limited. We used solid-state NMR (ssNMR) spectroscopy to deduce structural properties of both the A 36F 41A 46 mutant (AFA-PLN) and wild-type PLN (WT-PLN) when bound to SERCA1a after reconstitution in a functional lipid bilayer environment. Chemical-shift assignments in all domains of AFA-PLN provide direct evidence for the presence of two terminal alpha helices connected by a linker region of reduced structural order that differs from previous findings on free PLN. ssNMR experiments on WT-PLN show no significant difference in binding compared to AFA-PLN and do not support the coexistence of a significantly populated dynamic state of PLN after formation of the PLN/SERCA complex. A combination of our spectroscopic data with biophysical and biochemical data using flexible protein-protein docking simulations provides a structural basis for understanding the interaction between PLN and SERCA1a.

Complex Formation and Light Activation in Membrane-Embedded Sensory Rhodopsin II as Seen by Solid-State NMR Spectroscopy

Microbial rhodopsins execute diverse biological functions in the cellular membrane. A mechanistic understanding of their functional profile is, however, still limited. We used solid-state NMR (ssNMR) spectroscopy to study structure and dynamics of a 2 x 400 amino acid sensory rhodopsin/transducer (SRII/HtrII) complex from Natronomonas pharaonis in a natural membrane environment. We found a receptor-transducer binding interface in the ground state that significantly extends beyond the available X-ray structure. This binding domain involves the EF loop of the receptor and stabilizes the functionally relevant, directly adjacent HAMP domain of the transducer. Using 2D ssNMR difference spectroscopy, we identified protein residues that may act as a functional module around the retinal binding site during the early events of protein activation. These latter protein segments, the inherent plasticity of the HAMP domain, and the observation of an extended SRII/HtrII membrane-embedded interface may be crucial components for optimal signal relay efficiency across the cell membrane.

Protein solid-state NMR resonance assignments from (13C,13C) correlation spectroscopy

It is demonstrated that sequential resonance assignments can be obtained from (13C,13C) correlation spectroscopy on a uniformly labeled protein under magic angle spinning. The experiment relies on weak (C′,Cα) coupling conditions using a defined range of MAS rates and can be employed at arbitrary magnetic field strength.

Probing molecular motion by double-quantum (13C,13C) solid-state NMR spectroscopy: Application to ubiquitin

We demonstrate the use of two-dimensional ((13)C,(13)C) double-quantum spectroscopy to detect molecular dynamics by solid-state NMR. Data collected on tyrosine-ethylester (TEE) are in line with previously determined ((1)H,(13)C) order parameters. Application of these experiments to microcrystalline ubiquitin reveals the presence of dynamics on millisecond or faster time scales and differences in local mobility depending on microcrystal preparation. In addition, solid-state NMR-based structure calculation indicates conformational variability of loop regions between different solid-phase ubiquitin preparations. Our data relate preparation-dependent changes observed in NMR spectral parameters such as chemical shifts and through-space correlations to differences in ubiquitin dynamics and conformation and suggest a prominent role of molecular mobility in microcrystalline ubiquitin.

Ion distribution in copper exchanged zeolites by using Si-29 spin lattice relaxation analysis.

Transition metal-containing zeolites, particularly those with smaller pore size, have found extensive application in the selective catalytic reduction (SCR) of environmental pollutants containing nitrogen oxides. We report these zeolites have dramatically faster silicon-29 (Si-29) spin lattice relaxation times (T1) compared to their sodium-containing counterparts. Paramagnetic doping allows one to acquire Si-29 MAS spectra in the order of tens of seconds without significantly affecting the spectral resolution. Moreover, relaxation times depend on the method of preparation and the next-nearest neighbor silicon Qn(mAl) sites, where n=4 and m=0-4, respectively. A clear trend is noted between the effectiveness of Cu exchange and the Si-29 NMR relaxation times. It is anticipated that the availability of this tool, and the enhanced understanding of the nature of the active sites, will provide the means for designing improved SCR catalysts.