High Resolution Atomic Force Microscopy of Functional Biological Molecules

Nanoscale dynamic biological processes are central to the regulation of cellular pro- cesses within the body. The direct visualisation of these processes represents a challenge because of the intrinsic difficulties of imaging at the nanoscale, well below the diffraction limit of light. Here we use the Atomic Force Microscope to ‘feel’ the structure of single biomolecules adsorbed to a flat substrate at sub-nanometre resolution. We have enhanced the performance and resolution of Atomic Force Microscopy (AFM) for imaging DNA plasmids in solution, resolving its secondary structure in the form of the double helix. We are able to observe local deviations from the average structure, and in particular variations in the depth of the grooves in the double-stranded DNA which may be attributed to supercoiling of the DNA. Such local variations of the DNA double helix structure are important in mediating protein-DNA binding specificity and thus in regulating gene expression. We show preliminary data on DNA minicircles, which can be used as a synthetic system to study how supercoiling affects DNA structure and influences DNA-protein binding interactions with implications for many genetic processes. Going from fundamental science to a biomedical application, we have used AFM to study the functional mechanisms of antimicrobial peptides, which are developed in response to the growing problem of antimicrobial resistance. Antimicrobial peptides disrupt microbial phospholipid membranes but direct observation of the mode of action for the disruption is lacking. Here we visualise the mode of action of syn- thetic antimicrobial cationic alpha-helical peptides. Two of these peptides attack membrane via previously unknown mechanism: Amhelin forms pores which are not limited in size but expand from the nano to micrometre scale; Amhelit also forms pores which penetrate a single layer of the lipid bilayer that forms the membrane. We present the first nanoscale visualisation of membrane disruption by the naturally occurring antimicrobial peptide cecropin B. This is complemented by the visualisa- tion of peptides similar in sequence to cecropin B, but with structural modifications which are used to elucidate the structural origins of cecropin B’s mechanism of ac- tion. Improvements in imaging capabilities of the AFM, as tested on DNA, were shown to benefit imaging of the mode of action for antimicrobial peptides, including time-lapse imaging of a novel expanding monolayer state. We have thus used AFM to elucidate mechanisms of action for antimicrobial pep- tides. Relating these mechanisms to the peptide sequences, we can gain insight into how peptide sequence affects structure and function for these antimicrobial agents. This may aid in the development and improvement of novel peptide antibiotics

Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

Atomic force microscopy (AFM) provides an effective, label-free technique enabling the imaging of live bacteria under physiological conditions with nanometre precision. However, AFM is a surface scanning technique, and the accuracy of its performance requires the effective and reliable immobilisation of bacterial cells onto substrates. Here, we compare the effectiveness of various chemical approaches to facilitate the immobilisation of Escherichia coli onto glass cover slips in terms of bacterial adsorption, viability and compatibility with correlative imaging by fluorescence microscopy. We assess surface functionalisation using gelatin, poly-l-lysine, Cell-Tak™, and Vectabond®. We describe how bacterial immobilisation, viability and suitability for AFM experiments depend on bacterial strain, buffer conditions and surface functionalisation. We demonstrate the use of such immobilisation by AFM images that resolve the porin lattice on the bacterial surface; local degradation of the bacterial cell envelope by an antimicrobial peptide (Cecropin B); and the formation of membrane attack complexes on the bacterial membrane

PEGylated surfaces for the study of DNA-protein interactions by atomic force microscopy

DNA-protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA-protein interactions at nanometre resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working in biologically relevant protein concentrations, where proteins may be present in large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLLx-b-PEGy block copolymers to achieve selective adsorption of DNA on a mica surface for AFM studies. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, we show selective adsorption of DNA on mica that is functionalized with a PLL10-b-PEG113/PLL1000-2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. We show - through the use of biotinylated DNA and streptavidin - that this selective adsorption extends to DNA-protein complexes and that DNA-bound streptavidin can be unambiguously distinguished in spite of an excess of unbound streptavidin in solution. Finally, we apply this to the nuclear enzyme PARP1, resolving the binding of individual PARP1 molecules to DNA by in-liquid AFM

Imaging the Effects of Peptide Materials on Phospholipid Membranes by Atomic Force Microscopy

Recent advances in biomolecular design require accurate measurements performed in native or near-native environments in real time. Atomic force microscopy (AFM) is a powerful tool to observe the dynamics of biologically relevant processes at aqueous interfaces with high spatial resolution. Here, we describe imaging protocols to characterize the effects of peptide materials on phospholipid membranes in solution by AFM. These protocols can be used to determine the mechanism and kinetics of membrane-associated activities at the nanoscale

Biomimetic hybrid nanocontainers with selective permeability

Chemistry plays a crucial role in creating synthetic analogues of biomacromolecular structures. Of particular scientific and technological interest are biomimetic vesicles that are inspired by natural membrane compartments and organelles but avoid their drawbacks, such as membrane instability and limited control over cargo transport across the boundaries. In this study, completely synthetic vesicles were developed from stable polymeric walls and easy‐to‐engineer membrane DNA nanopores. The hybrid nanocontainers feature selective permeability and permit the transport of organic molecules of 1.5 nm size. Larger enzymes (ca. 5 nm) can be encapsulated and retained within the vesicles yet remain catalytically active. The hybrid structures constitute a new type of enzymatic nanoreactor. The high tunability of the polymeric vesicles and DNA pores will be key in tailoring the nanocontainers for applications in drug delivery, bioimaging, biocatalysis, and cell mimicry