Andreas Hoenger

De novo designed peptide-based amyloid fibrils

Identification of therapeutic strategies to prevent or cure diseases associated with amyloid fibril deposition in tissue (Alzheimers disease, spongiform encephalopathies, etc.) requires a rational understanding of the driving forces involved in the formation of these organized assemblies rich in β-sheet structure. To this end, we used a computer-designed algorithm to search for hexapeptide sequences with a high propensity to form homopolymeric β-sheets. Sequences predicted to be highly favorable on this basis were found experimentally to self-associate efficiently into β-sheets, whereas point mutations predicted to be unfavorable for this structure inhibited polymerization. However, the property to form polymeric β-sheets is not a sufficient requirement for fibril formation because, under the conditions used here, preformed β-sheets from these peptides with charged residues form well defined fibrils only if the total net charge of the molecule is ±1. This finding illustrates the delicate balance of interactions involved in the formation of fibrils relative to more disordered aggregates. The present results, in conjunction with x-ray fiber diffraction, electron microscopy, and Fourier transform infrared measurements, have allowed us to propose a detailed structural model of the fibrils.

Correlative microscopy and electron tomography of GFP through photooxidation

We have developed a simple correlative photooxidation method that allows for the direct ultrastructural visualization of the green fluorescent protein (GFP) upon illumination. The method, termed GRAB for GFP recognition after bleaching, uses oxygen radicals generated during the GFP bleaching process to photooxidize 3,3′-diaminobenzidine (DAB) into an electron-dense precipitate that can be visualized by routine electron microscopy and electron tomography. The amount of DAB product produced by the GRAB method appears to be linear with the initial fluorescence, and the resulting images are of sufficient quality to reveal detailed spatial information. This is exemplified by the observed intra–Golgi stack and intracisternal distribution of a human Golgi resident glycosylation enzyme, N-acetylgalactosaminyltransferase-2 fused either to enhanced GFP or CFP.

A 3D analysis of yeast ER structure reveals how ER domains are organized by membrane curvature

Electron tomography of continuous ER domains during budding shows that reticulons and Yop1 stabilize rather than generate membrane curvature in this organelle.

A Model for the Microtubule-Ncd Motor Protein Complex Obtained by Cryo-Electron Microscopy and Image Analysis

Kinesin motors convert chemical energy from ATP hydrolysis into unidirectional movement. To understand how kinesin motors bind to and move along microtubules, we fit the atomic structure of the motor domain of Ncd (a kinesin motor involved in meiosis and mitosis) into three-dimensional density maps of Ncd-microtubule complexes calculated by cryo-electron microscopy and image analysis. The model reveals that Ncd shares an extensive interaction surface with the microtubule, and that a portion of the binding site involves loops that contain conserved residues. In the Ncd dimer, the microtubule-bound motor domain makes intimate contact with its partner head, which is dissociated from the microtubule. This head-head interaction may be important in positioning the dissociated head to take a step to the next binding site on the microtubule protofilament.

The Schizosaccharomyces pombe EB1 Homolog Mal3p Binds and Stabilizes the Microtubule Lattice Seam

End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing we have studied the microtubule-binding properties of the fission yeast EB1 homolog Mal3p. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. We provide structural data showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam as well as data suggesting that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. Our findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes.

Axial Stretch of Rat Single Ventricular Cardiomyocytes Causes an Acute and Transient Increase in Ca2+ Spark Rate

We investigate acute effects of axial stretch, applied by carbon fibers (CFs), on diastolic Ca2+ spark rate in rat isolated cardiomyocytes. CFs were attached either to both cell ends (to maximize the stretched region), or to the center and one end of the cell (to compare responses in stretched and nonstretched half-cells). Sarcomere length was increased by 8.01±0.94% in the stretched cell fraction, and time series of XY confocal images were recorded to monitor diastolic Ca2+ spark frequency and dynamics. Whole-cell stretch causes an acute increase of Ca2+ spark rate (to 130.7±6.4%) within 5 seconds, followed by a return to near background levels (to 104.4±5.1%) within 1 minute of sustained distension. Spark rate increased only in the stretched cell region, without significant differences in spark amplitude, time to peak, and decay time constants of sparks in stretched and nonstretched areas. Block of stretch-activated ion channels (2 &mgr;mol/L GsMTx-4), perfusion with Na+/Ca2+-free solution, and block of nitric oxide synthesis (1 mmol/L L-NAME) all had no effect on the stretch-induced acute increase in Ca2+ spark rate. Conversely, interference with cytoskeletal integrity (2 hours of 10 &mgr;mol/L colchicine) abolished the response. Subsequent electron microscopic tomography confirmed the close approximation of microtubules with the T-tubular–sarcoplasmic reticulum complex (to within ≈10-8m). In conclusion, axial stretch of rat cardiomyocytes acutely and transiently increases sarcoplasmic reticulum Ca2+ spark rate via a mechanism that is independent of sarcolemmal stretch-activated ion channels, nitric oxide synthesis, or availability of extracellular calcium but that requires cytoskeletal integrity. The potential of microtubule-mediated modulation of ryanodine receptor function warrants further investigation.

GTPgammaS microtubules mimic the growing microtubule end structure recognized by end-binding proteins (EBs)

Microtubule plus-end-tracking proteins (+TIPs) localize to growing microtubule plus ends to regulate a multitude of essential microtubule functions. End-binding proteins (EBs) form the core of this network by recognizing a distinct structural feature transiently existing in an extended region at growing microtubule ends and by recruiting other +TIPs to this region. The nature of the conformational difference allowing EBs to discriminate between tubulins in this region and other potential tubulin binding sites farther away from the microtubule end is unknown. By combining in vitro reconstitution, multicolor total internal reflection fluorescence microscopy, and electron microscopy, we demonstrate here that a closed microtubule B lattice with incorporated GTPγS, a slowly hydrolyzable GTP analog, can mimic the natural EB protein binding site. Our findings indicate that the guanine nucleotide γ-phosphate binding site is crucial for determining the affinity of EBs for lattice-incorporated tubulin. This defines the molecular mechanism by which EBs recognize growing microtubule ends.

CTF determination and correction for low dose tomographic tilt series

The resolution of cryo-electron tomography can be limited by the first zero of the microscopes contrast transfer function (CTF). To achieve higher resolution, it is critical to determine the CTF and correct its phase inversions. However, the extremely low signal-to-noise ratio (SNR) and the defocus gradient in the projections of tilted specimens make this process challenging. Two programs, CTFPLOTTER and CTFPHASEFLIP, have been developed to address these issues. CTFPLOTTER obtains a 1D power spectrum by periodogram averaging and rotational averaging and it estimates the noise background with a novel approach, which uses images taken with no specimen. The background-subtracted 1D power spectra from image regions at different defocus values are then shifted to align their first zeros and averaged together. This averaging improves the SNR sufficiently that it becomes possible to determine the defocus for subsets of the tilt series rather than just the entire series. CTFPHASEFLIP corrects images line-by-line by inverting phases appropriately in thin strips of the image at nearly constant defocus. CTF correction by these methods is shown to improve the resolution of aligned, averaged particles extracted from tomograms. However, some restoration of Fourier amplitudes at high frequencies is important for seeing the benefits from CTF correction.

Microscopic evidence for a minus‐end‐directed power stroke in the kinesin motor ncd

We used cryo‐electron microscopy and image reconstruction to investigate the structure and microtubule‐binding configurations of dimeric non‐claret disjunctional (ncd) motor domains under various nucleotide conditions, and applied molecular docking using ncds dimeric X‐ray structure to generate a mechanistic model for force transduction. To visualize the α‐helical coiled‐coil neck better, we engineered an SH3 domain to the N‐terminal end of our ncd construct (296–700). Ncd exhibits strikingly different nucleotide‐dependent three‐dimensional conformations and microtubule‐binding patterns from those of conventional kinesin. In the absence of nucleotide, the neck adapts a configuration close to that found in the X‐ray structure with stable interactions between the neck and motor core domain. Minus‐end‐directed movement is based mainly on two key events: (i) the stable neck–core interactions in ncd generate a binding geometry between motor and microtubule which places the motor ahead of its cargo in the minus‐end direction; and (ii) after the uptake of ATP, the two heads rearrange their position relative to each other in a way that promotes a swing of the neck in the minus‐end direction.

Structure of a fast kinesin: implications for ATPase mechanism and interactions with microtubules

We determined the crystal structure of the motor domain of the fast fungal kinesin from Neurospora crassa (NcKin). The structure has several unique features. (i) Loop 11 in the switch 2 region is ordered and enables one to describe the complete nucleotide‐binding pocket, including three inter‐switch salt bridges between switch 1 and 2. (ii) Loop 9 in the switch 1 region bends outwards, making the nucleotide‐binding pocket very wide. The displacement in switch 1 resembles that of the G‐protein ras complexed with its guanosine nucleotide exchange factor. (iii) Loop 5 in the entrance to the nucleotide‐binding pocket is remarkably long and interacts with the ribose of ATP. (iv) The linker and neck region is not well defined, indicating that it is mobile. (v) Image reconstructions of ice‐embedded microtubules decorated with NcKin show that it interacts with several tubulin subunits, including a central β‐tubulin monomer and the two flanking α‐tubulin monomers within the microtubule protofilament. Comparison of NcKin with other kinesins, myosin and G‐proteins suggests that the rate‐limiting step of ADP release is accelerated in the fungal kinesin and accounts for the unusually high velocity and ATPase activity.