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High Resolution Atomic Force Microscopy of Functional Biological Molecules

Pyne, ALB; (2015) High Resolution Atomic Force Microscopy of Functional Biological Molecules. Doctoral thesis , UCL (University College London). Green open access

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Abstract

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.

Type: Thesis (Doctoral)
Title: High Resolution Atomic Force Microscopy of Functional Biological Molecules
Event: University College London
Open access status: An open access version is available from UCL Discovery
Language: English
Keywords: Atomic Force Microscopy, Biophysics, DNA, Antimicrobial Peptides, Antimicrobial Resistance, Nanoscience
UCL classification: UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > London Centre for Nanotechnology
URI: http://discovery.ucl.ac.uk/id/eprint/1470215
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