Kenneth N. Goldie

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.

Scan speed limit in atomic force microscopy

The scan speed limit of atomic force microscopes has been calculated. It is determined by the spring constant of the cantilever k, its effective mass m, the damping constant D of the cantilever in the surrounding medium and the stiffness of the sample. Techniques to measure k, k/m and D/m are described. In liquids the damping constant and the effective mass of the cantilever increase. A consequence of this is that the transfer function always depends on the scan speed when imaging in liquids. The practical scan speed limit for atomic resolution in vacuum is 0·1 μm/s while in water it increases to about 2 μm/s due to the additional damping of cantilever movements. Sample stiffness or damping of cantilever movements by the sample increase these limits. For soft biological materials imaged in water at a desired resolution of 1 nm the scan speed should not exceed 2 μm/s.

Factors influencing the precision of quantitative scanning transmission electron microscopy

Abstract The scanning transmission electron microscope (STEM) can be used for accurate and reproducible mass measurements. Here we analyse the major sources of systematic errors. Focus-dependent changes of the magnification can be corrected on-line by monitoring the objective-lens current. Post-specimen field effects are shown to be negligible for the Vacuum Generators STEM HB5 used. Operating conditions of the detector, a scintillator-photomultiplier combination, are critical and need to be calibrated for each experiment. The influence of sample purity, mass-loss kinetics and glutaraldehyde fixation on mass values is evaluated for several biological specimens, in particular for the widely used mass standard TMV. Possible errors arising from the use of mass standards to compensate for both instrumental and specimen-related uncertainties are considered.

Capsosomes: Subcompartmentalizing Polyelectrolyte Capsules Using Liposomes

Next-generation therapeutic approaches are expected to rely on the engineering of multifunctional particle carriers that can mimic specific cellular functions. The key features of such particles are the semipermeable nature of the shell for communication with the external environment and multiple nanosized individual subcompartments confined within a micron-sized structurally stable scaffold for conducting specific reactions. Herein, we report the formation of capsosomes, a new class of polyelectrolyte capsules containing structurally intact liposomes as cargo. The multilayer film assembly of polyelectrolytes (poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH)) and liposomes (50 nm 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) was characterized on planar substrates using quartz crystal microbalance with dissipation monitoring, and these findings were then correlated to the film growth of the polyelectrolytes and structurally intact liposomes on silica particles. Upon removal of the silica template core, stable capsosomes, containing one or two layers of intact liposomes as cargo, were obtained. This novel platform, capsosomes, which combines the advantages of two systems, liposomes and polyelectrolyte capsules, is expected to find diverse applications in biomedicine, in particular for the creation of artificial cells or organelles where the performance of reactions within a confined environment is a prerequisite.

Op18/stathmin caps a kinked protofilament‐like tubulin tetramer

Oncoprotein 18/stathmin (Op18), a regulator of microtubule dynamics, was recombinantly expressed and its structure and function analysed. We report that Op18 by itself can fold into a flexible and extended α‐helix, which is in equilibrium with a less ordered structure. In complex with tubulin, however, all except the last seven C‐terminal residues of Op18 are tightly bound to tubulin. Digital image analysis of Op18:tubulin electron micrographs revealed that the complex consists of two longitudinally aligned α/β‐tubulin heterodimers. The appearance of the complex was that of a kinked protofilament‐like structure with a flat and a ribbed side. Deletion mapping of Op18 further demonstrated that (i) the function of the N‐terminal part of the molecule is to ‘cap’ tubulin subunits to ensure the specificity of the complex and (ii) the complete C‐terminal α‐helical domain of Op18 is necessary and sufficient for stable Op18:tubulin complex formation. Together, our results suggest that besides sequestering tubulin, the structural features of Op18 enable the protein specifically to recognize microtubule ends to trigger catastrophes.

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.

Assembly of Poly(dopamine) Films Mixed with a Nonionic Polymer

Poly(dopamine) (PDA) coatings have recently attracted considerable interest for a variety of applications. Here, we investigate the film deposition of dopamine mixed with a nonionic polymer (i.e., poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), and poly(N-vinyl pyrrolidone) (PVP)) onto silica substrates using X-ray photoelectron spectroscopy and quartz crystal microbalance. Furthermore, we assess the possibility of coating silica colloids to yield polymer capsules and liposomes with these mixtures. We found that mixed PDA/PEG and PDA/PVA films are deposited without the need for a covalent linker such as an amine or thiol. We also discovered the first material, namely, PVP, that can suppress PDA film assembly. These fundamental findings give further insight into PDA film properties and contribute to establish PDA as a widely applicable coating.

Structure of the T4 baseplate and its function in triggering sheath contraction.

Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail. We establish the identity and function of a minimal set of components that is conserved in all contractile injection systems and show that the triggering mechanism is universally conserved.

Functional significance of symmetrical versus asymmetrical GroEL-GroES chaperonin complexes

The Escherichia coli chaperonin GroEL and its regulator GroES are thought to mediate adenosine triphosphate-dependent protein folding as an asymmetrical complex, with substrate protein bound within the GroEL cylinder. In contrast, a symmetrical complex formed between one GroEL and two GroES oligomers, with substrate protein binding to the outer surface of GroEL, was recently proposed to be the functional chaperonin unit. Electron microscopic and biochemical analyses have now shown that unphysiologically high magnesium concentrations and increased pH are required to assemble symmetrical complexes, the formation of which precludes the association of unfolded polypeptide. Thus, the functional significance of GroEL:(GroES)2 particles remains to be demonstrated.

Visualization of cell microtubules in their native state

Background information. Over the past decades, cryo‐electron microscopy of vitrified specimens has yielded a detailed understanding of the tubulin and microtubule structures of samples reassembled in vitro from purified components. However, our knowledge of microtubule structure in vivo remains limited by the chemical treatments commonly used to observe cellular architecture using electron microscopy.