Bart W. Hoogenboom

Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers

Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.

Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin

Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing. DOI: http://dx.doi.org/10.7554/eLife.04247.001

Imaging Surface Charges of Individual Biomolecules

Surface charges play a key role in determining the structure and function of proteins, DNA, and larger biomolecular structures. Here we report on the measurement of the electrostatic surface potential of individual DNA and avidin molecules with nanometer resolution using Kelvin probe force microscopy. We also show, for the first time, the surface potential of buffer salts shielding individual DNA molecules, which would not be possible with conventional ensemble techniques.

Atomic Force Microscopy with Nanoscale Cantilevers Resolves Different Structural Conformations of the DNA Double Helix

Structural variability and flexibility are crucial factors for biomolecular function. Here we have reduced the invasiness and enhanced the spatial resolution of atomic force microscopy (AFM) to visualize, for the first time, different structural conformations of the two polynucleotide strands in the DNA double helix, for single molecules under near-physiological conditions. This is achieved by identifying and tracking the anomalous resonance behavior of nanoscale AFM cantilevers in the immediate vicinity of the sample.

Single-molecule reconstruction of oligonucleotide secondary structure by atomic force microscopy.

Based on soft-touch atomic force microscopy, a method is described to reconstruct the secondary structure of single extended biomolecules, without the need for crystallization. The method is tested by accurately reproducing the dimensions of the B-DNA crystal structure. Importantly, intramolecular variations in groove depth of the DNA double helix are resolved, which would be inaccessible for methods that rely on ensemble-averaging.

Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes

The nuclear pore complex (NPC) is the gate for transport between the cell nucleus and the cytoplasm. Small molecules cross the NPC by passive diffusion, but molecules larger than ~5 nm must bind to nuclear transport receptors to overcome a selective barrier within the NPC1. Whilst the structure and shape of the cytoplasmic ring of the NPC are relatively well characterized2-5, the selective barrier is situated deep within the central channel of the NPC and depends critically on unstructured nuclear pore proteins5,6, and is therefore not well understood. Here, we show that stiffness topography7 with sharp atomic force microscopy tips can generate nanoscale cross sections of the NPC. The cross sections reveal two distinct structures, a cytoplasmic ring and a central plug structure, which are consistent with the three-dimensional NPC structure derived from electron microscopy2-5. The central plug persists after reactivation of the transport cycle and resultant cargo release, indicating that the plug is an intrinsic part of the NPC barrier. Added nuclear transport receptors accumulate on the intact transport barrier and lead to a homogenization of the barrier stiffness. The observed nanomechanical properties in the NPC indicate the presence of a cohesive barrier to transport, and are quantitatively consistent with the presence of a central condensate of nuclear pore proteins in the NPC channel.

Improved Kelvin probe force microscopy for imaging individual DNA molecules on insulating surfaces

Electrostatic forces and potentials are keys in determining the interactions between biomolecules. We have recently imaged the topography and electrostatic surface potential of nucleic acid molecules on silicon surfaces using Kelvin probe force microscopy (KPFM). Here, we demonstrate KPFM imaging on insulating surfaces like mica, which provides access to configurations of DNA that are projections of its structure in solution. In particular, we apply dual-frequency mode to minimize the tip-sample distance at which the Kelvin probe signal is acquired and use the fundamental resonance of the cantilever to determine surface potential and its first overtone to detect the topography.

Bistable collective behavior of polymers tethered in a nanopore.

Polymer-coated pores play a crucial role in nucleo-cytoplasmic transport and in a number of biomimetic and nanotechnological applications. Here we present Monte Carlo and Density Functional Theory approaches to identify different collective phases of end-grafted polymers in a nanopore and to study their relative stability as a function of intermolecular interactions. Over a range of system parameters that is relevant for nuclear pore complexes, we observe two distinct phases: one with the bulk of the polymers condensed at the wall of the pore, and the other with the polymers condensed along its central axis. The relative stability of these two phases depends on the interpolymer interactions. The existence the two phases suggests a mechanism in which marginal changes in these interactions, possibly induced by nuclear transport receptors, cause the pore to transform between open and closed configurations, which will influence transport through the pore.

A physical model describing the interaction of nuclear transport receptors with FG nucleoporin domain assemblies

The permeability barrier of nuclear pore complexes (NPCs) controls bulk nucleocytoplasmic exchange. It consists of nucleoporin domains rich in phenylalanine-glycine motifs (FG domains). As a bottom-up nanoscale model for the permeability barrier, we have used planar films produced with three different end-grafted FG domains, and quantitatively analyzed the binding of two different nuclear transport receptors (NTRs), NTF2 and Importin β, together with the concomitant film thickness changes. NTR binding caused only moderate changes in film thickness; the binding isotherms showed negative cooperativity and could all be mapped onto a single master curve. This universal NTR binding behavior – a key element for the transport selectivity of the NPC – was quantitatively reproduced by a physical model that treats FG domains as regular, flexible polymers, and NTRs as spherical colloids with a homogeneous surface, ignoring the detailed arrangement of interaction sites along FG domains and on the NTR surface. DOI: http://dx.doi.org/10.7554/eLife.14119.001

Disentangling mechanical and mass effects on nanomechanical resonators

Micro and nanomechanical resonators are powerful and label-free sensors of analytes in various environments. Their response, however, is a convolution of mass, rigidity, and nanoscale heterogeneity of adsorbates. Here we demonstrate a procedure to disentangle this complex sensor response, to simultaneously measure both mass and elastic properties of nanometer thick samples. This turns an apparent disadvantage of these resonators into a striking and unique asset, enabling them to measure more than mass alone.