Massimo Antognozzi

Self-assembling cages from coiled-coil peptide modules

From Coils to Cages Self-assembly strategies that mimic protein assembly, such as the formation of viral coats, often begin with simpler peptide assemblies. Fletcher et al. (p. 595, published online 11 April; see the Perspective by Ardejani and Orner) designed two coiled-coil peptide motifs, a heterodimer, and a homotrimer. Both peptides contained cysteine residues and could link through disulfide bonds, so that the trimer could form the vertices of a hexagonal network and the dimer its edges. However, these components are flexible and, rather than form extended sheets, they closed to form particles ∼100 nanometers in diameter. Hexagonal networks form from heterodimeric and homotrimeric coiled coils and create ~100-nanometer-diameter cages. [Also see Perspective by Ardejani and Orner] An ability to mimic the boundaries of biological compartments would improve our understanding of self-assembly and provide routes to new materials for the delivery of drugs and biologicals and the development of protocells. We show that short designed peptides can be combined to form unilamellar spheres approximately 100 nanometers in diameter. The design comprises two, noncovalent, heterodimeric and homotrimeric coiled-coil bundles. These are joined back to back to render two complementary hubs, which when mixed form hexagonal networks that close to form cages. This design strategy offers control over chemistry, self-assembly, reversibility, and size of such particles.

Breaking the speed limit with atomic force microscopy

High-speed atomic force microscopy (AFM) is important for following processes that occur on sub-second timescales for studies both in biology and materials science, and also for the ability to examine large areas of a specimen at high resolution in a practical length of time. Further developments of the previously reported high-speed contact-mode AFM are described. Two instruments are presented: (i) a high-speed flexure stage arrangement capable of imaging at a video rate of 30 fps, and (ii) an ultra-high speed instrument using a combined tuning fork and flexure-stage scanning system capable of ultra-high-speed imaging in excess of 1000 fps. Results of imaging collagen fibres under ambient conditions at rates of up to 1300 frames s−1 are presented. Despite tip–specimen relative velocities of up to 200 mm s−1, no significant damage to the collagen specimen was observed even after tens of thousands of frames were acquired in the same area of the specimen.

Observation of molecular layering in a confined water film and study of the layers viscoelastic properties

A transverse dynamic force microscope, more commonly known as shear force microscope, has been used to investigate confined water films under shear. A cylindrically tapered glass probe was mounted perpendicularly to the sample surface. Pure water was confined between the probe and a freshly cleaved mica surface and a sinusoidal shear strain was applied by setting the probe into transverse oscillation. Repeated measurements of the probe oscillation amplitude and relative phase lag, at different tip-sample separations, exhibited a clear step-like behavior. The periodicity, recorded over several curves, ranged between 2.4 and 2.9 A, which is similar to the diameter of the water molecule. The in-phase (elastic) and the out-of-phase (viscous) stress response of the confined water film was evaluated (from the experimental data) by assuming a linear viscoelastic behavior. Finally, by modeling the water film with the Maxwell mechanical model, the values for the shear viscosity and shear rigidity were obtained.

A chlorite mineral surface actively drives the deposition of DNA molecules in stretched conformations

Muscovite mica is commonly used to immobilize DNA molecules onto a flat surface. This method, however, requires either the use of divalent cations in the buffer solution or the chemical modification of the surface. Here we show that DNA molecules have different binding affinities and assume different conformations when adsorbed to different layered minerals. In particular, the effect of biotite, muscovite, talc, brucite and chlorite upon DNA binding is investigated. Using atomic force microscopy it is possible to quantify the amount of DNA deposited onto a flat surface and it is experimentally confirmed that biotite, talc and brucite have a much higher affinity than muscovite (7-, 20- and 25-fold more volume of DNA deposited, respectively). The deposition of DNA onto chlorite presents areas (brucite-like) with high DNA coverage and areas (mica-like) where DNA molecules are absent. We regularly observed isolated DNA molecules that became stretched across these regions of low affinity. The stretching is not induced by the deposition procedure but is driven by the surface potential gradient between brucite-like and mica-like regions in chlorite. The active stretching of DNA on chlorite is a clear indication of the technological potential carried by these materials when used as substrates for biomolecules.

Shear response of nanoconfined water on muscovite mica: role of cations.

By monitoring the thermal noise of a vertically oriented micromechanical force sensor, we detect the viscoelastic response to shear for water in a subnanometer confinement. Measurements in pure water as well as under acidic and high-ionic-strength conditions relate this response to the effect of surface-adsorbed cations, which, because of their hydration, act as pinning centers restricting the mobility of the confined water molecules.

Small-molecule uptake in membrane-free peptide/nucleotide protocells

The spontaneous phase separation of peptide/nucleotide droplets in water produces membrane-free chemically organized micro-compartments that offer new opportunities for the construction of synthetic cells and development of protocell models of prebiotic organization. Certain small molecules can be sequestered into the droplet interior but the uptake mechanisms are unexplored. Using confocal fluorescence microscopy, 31P NMR spectroscopy, fluorescence spectroscopy and lateral molecular force microscopy, we probe the molecular interactions associated with sequestration of the water-soluble fluorescent anionic dye 1-anilinonapthalene-8-sulphonic acid (ANS) into positively charged oligolysine/ATP coacervate micro-droplets. Our results indicate that uptake of ANS proceeds initially through electrostatic interactions involving a ternary ANS/oligolysine/ATP complex, followed by a secondary mechanism based on non-polar interactions between ANS and ATP. We demonstrate that at very high levels of ANS the hybrid droplets develop a thin outer shell that is mechanically more compliant than the droplet interior, and acts as a quasi-membrane for restricting the influx of methylene blue. Our results suggest that understanding the mechanisms of molecular uptake into coacervate droplets could provide an important step towards the rational design of molecularly crowded microscale dispersions that display complex fluid behavior, compartment-mediated functionality and primitive aspects of synthetic cellularity.

Correlation of in situ mechanosensitive responses of the Moraxella catarrhalis adhesin UspA1 with fibronectin and receptor CEACAM1 binding

Bacterial cell surfaces are commonly decorated with a layer formed from multiple copies of adhesin proteins whose binding interactions initiate colonization and infection processes. In this study, we investigate the physical deformability of the UspA1 adhesin protein from Moraxella catarrhalis, a causative agent of middle-ear infections in humans. UspA1 binds a range of extracellular proteins including fibronectin, and the epithelial cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Electron microscopy indicates that unliganded UspA1 is densely packed at, and extends about 800 Å from, the Moraxella surface. Using a modified atomic force microscope, we show that the adhesive properties and thickness of the UspA1 layer at the cell surface varies on addition of either fibronectin or CEACAM1. This in situ analysis is then correlated with the molecular structure of UspA1. To provide an overall model for UspA1, we have determined crystal structures for two N-terminal fragments which are then combined with a previous structure of the CEACAM1-binding site. We show that the UspA1–fibronectin complex is formed between UspA1 head region and the 13th type-III domain of fibronectin and, using X-ray scattering, that the complex involves an angular association between these two proteins. In combination with a previous study, which showed that the CEACAM1–UspA1 complex is distinctively bent in solution, we correlate these observations on isolated fragments of UspA1 with its in situ response on the cell surface. This study therefore provides a rare direct demonstration of protein conformational change at the cell surface.

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times of Cy3-EDA-ATP (2′(3′)-O-[[2-[[6-[2-[3-(1-ethyl-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-5-sulfo-3H-indolio]-1-oxohexyl]amino]ethyl]carbamoyl]ATP) but reduced the number of Cy3-signals per field of view, approaching 50% at phosphate concentrations of 1–2 mm. Apparently, inhibition of movement does not result from binding of phosphate to an actomyosin-ADP intermediate as proposed by Hooft and coworkers (Hooft, A. M., Maki, E. J., Cox, K. K., and Baker, J. E. (2007) Biochemistry 46, 3513–3520) but, rather, from forming a strong-binding actomyosin-phosphate intermediate.

Processive behaviour of kinesin observed using micro-fabricated cantilevers.

The mechanical characterization of biomolecular motors requires force sensors with sub-piconewton resolution. The coupling of a nanoscale motor to this type of microscale sensors introduces structural deformations in the motor according to the thermally activated degrees of freedom of the sensor. At present, no simple solution is available to reduce these effects. Here, we exploit the advantages of micro-fabricated cantilevers to produce a force sensor with essentially one degree of freedom and a spring constant of 0.03 pN nm(-1) for the study of the molecular motor protein kinesin-1. During processive runs, the cantilever constrains the movement of the cargo binding domain of kinesin in a straight line, parallel to the microtubule track, and excludes specific reaction coordinates such as cargo rotation. In these conditions, we measured a step size of 8.0 ± 0.4 nm and a maximal unloaded velocity of 820 ± 80 nm s(-1) at saturated adenosine triphosphate (ATP) concentration. We concluded that the motor does not need to rotate its tail as it moves through consecutive stepping cycles.

Modeling of cylindrically tapered cantilevers for transverse dynamic force microscopy (TDFM).

In transverse dynamic force microscopy a cylindrically tapered cantilever is mounted perpendicularly to the sample surface and set into transversal oscillation. The dynamics of the cantilever has been studied using the continuum mechanical model with discrete element analysis. A viscoelastic model has been used to describe the tip-sample interaction. In this way an in-phase and an out-of-phase component of the force has been extracted from the experimental data. Two different techniques, involving two experimental setups and two corresponding data analysis routines, have been developed to calculate the two components of the force at different tip-sample separations. In one case the change in resonant frequency and corresponding oscillation amplitude is measured whereas in the second case the usual way of recording amplitude and phase signal at a fixed driving frequency is applied. The results from these two methods are shown to be completely consistent and produce almost identical force curves.