Fast and Angström-resolution AFM to visualise conformational change in biomolecules

Lead Research Organisation: University College London
Department Name: London Centre for Nanotechnology

Abstract

Atomic Force Microscopy (AFM) basically acts as a miniature blind man's stick ('cantilever') following the contours of a sample surface, and line by line reconstructing a three-dimensional representation of the surface topography. This line-by-line scanning is a fundamental difference from other, more common microscopy techniques and a main reason why it generally takes minutes to complete a single image. AFM is unique in combining sub-molecular resolution imaging with the ability to operate in liquids. For high-resolution imaging of biological samples, molecules are generally adsorbed on a hard surface, which is the only compromise compared to physiological conditions. Membrane proteins are samples of particular interest, since they represent more than 50% of modern drug targets and therefore are of major pharmaceutical importance. Their function as molecular nanomachines is determined by Angstrom-sized structural ('conformational') changes occurring at millisecond time scales. For applications in future healthcare and for basic scientific understanding, the crucial question is how molecular structure and changes in this structure relate to the biological function of membrane proteins. This project combines high-resolution AFM techniques (that have yielded atomic resolution!) with fast scanning, to obtain images of membrane proteins with Angstrom spatial and millisecond temporal resolution. This will enable us to visualise conformational changes in real time and observe biomolecules at work. This will be demonstrated on bacteriorhodopsin, a light-driven molecular machine that pumps protons through the cell membrane.

Technical Summary

The function of biomolecular machinery is determined by Angstrom-sized motion at millisecond time scales. This proposal outlines a development that will combine Angstrom-resolution atomic force microscopy (AFM) with fast scanning technology. The aim is real-time visualisation of Angstrom-sized conformational changes in membrane proteins under physiological conditions. This will be demonstrated by measuring the conformational changes that determine the direction of proton pumping in bacteriorhodopsin. The technique will be readily applied to the study of other biomolecular machines. First attempts to develop fast AFM date back to at least a decade ago. Several separate components necessary for fast and high-resolution AFM, however, have only been completed recently, and are yet to be integrated within an approach that yields Angstrom resolution at conventional scan speeds to start with. In particular, the highest-resolution operation modes at conventional speeds --- contact mode and FM-AFM, with accurate control of the tip-sample distance --- are yet to be implemented at high scan speeds. This will be the main instrumental innovation of this work. In addition, the scan-line triggered illumination will yield a much higher effective time resolution than can be achieved by monitoring molecules frame-by-frame only. Success in this direction depends on miniaturisation of cantilevers and on the ability to measure their deflections with highest sensitivity, such as can be achieved by the applicant's home-built deflection sensor.

Publications

10 25 50
 
Title Film The Demiurge inspired by our research 
Description Inspired by our research on imaging DNA by high-resolution atomic force microscopy, British artists AL and AL made a ski-fi artistic film, named The Demiurge, which has its world premiere February 2016 in a widely attended artistic exhibition in Manchester, and which was co-funded by the Wellcome Trust. 
Type Of Art Film/Video/Animation 
Year Produced 2016 
Impact Over 4000 visitors in first week of exhibition. 
URL http://homemcr.org/exhibition/al-al-incidents-of-travel-in-the-multiverse/
 
Description The ability to visualise single biological molecules in their native environment (salty water) at high resolution with probes that facilitate real-time imaging of biomolecular motion. This is exemplified by our observation, for the first time, of the DNA double helix on a single molecule in water.
Exploitation Route Characterisation of biomolecular surfaces in pharmaceutical industry. Collaborative agreements with atomic force microscopy manufacturers JPK Instruments and Bruker AXS, as well as a patent application that is presently being managed by UCL Business
Sectors Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Research methodology and technology developed in this project has benefitted industrial developments at Synganta and MedImmune (branch of AstraZeneca)
First Year Of Impact 2012
Sector Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description BBRSC CASE PhD studentship
Amount £102,000 (GBP)
Funding ID BB/M503113/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description BBSRC ALERT
Amount £173,000 (GBP)
Funding ID BB/R000042/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2017 
End 08/2018
 
Description Dynamics and pathways of assembly in membrane pore formation
Amount £403,245 (GBP)
Funding ID BB/J006254/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2012 
End 04/2015
 
Description EPSRC Equipment Funding
Amount £711,385 (GBP)
Funding ID EP/M028100/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 04/2015 
End 03/2016
 
Description Impact Acceleration Account
Amount £10,000 (GBP)
Funding ID BB/IAA/UCL/15 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2015 
End 09/2016
 
Description Wellcome Trust Small Arts Award
Amount £30,000 (GBP)
Funding ID 102626/Z/13/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2013 
End 03/2016
 
Title High-resolution atomic force microscopy 
Description Atomic force microscopy techniques to visualise DNA / nucleic acid / oligonucleotidedouble helix structure 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact Related work has benefitted industrial research by MedImmune (branch of AstraZeneca) and Syngenta. 
 
Description Imperial College London 
Organisation Imperial College London
Department Imperial College Trust
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Based on our research on the biological nuclear pore complex, we have proposed mechanisms via which artificial nanopores may be designed as selective valves. With collaborators at Imperial College, we currently attempt to implement these ideas in a working system. On a different topic, our work on pore forming proteins has helped us to initiate a collaboration with structural biologists at Imperial College, where we attempt to use similar methodology to elucidate mechanism via which the immune system attacks bacteria. We have now also started to collaborate on the formation of quadruply stranded DNA structures, as targeted by potential anticancer agents.
Collaborator Contribution Nanopore devices. Purified proteins. DNA constructs. Expertise.
Impact This is a multi-disciplinary collaboration, involving physicists, chemists, engineers and biologist. It is still rather early to highlight particular outcomes.
Start Year 2014
 
Description Industrial collaboration with AFM manufacturer Bruker Nano 
Organisation Bruker Corporation
Department Bruker Nano
Country Germany 
Sector Private 
PI Contribution Following successful high-resolution atomic force microscopy (AFM) imaging, we have signed a joint development agreement with world-leading AFM manufacturer Bruker Nano (formerly Veeco, formerly Digital Instruments), on testing and developing protocols on prototype AFM equipment. Bruker Nano contributes in-kind to this project. Our work with their instrumentation and probes has provided Bruker with AFM images of the DNA double helix (and protocols for acquiring these), as well as an assessment of probe tip sharpness (both by benchmarking on DNA and on assemblies of pore forming proteins.
Collaborator Contribution Provision of and access to latest commercial AFM equipment (including beta-version pre market release), provision of AFM proves, all at zero or greatly reduced price.
Impact Multidisciplinary - involving engineering, physics and biology. Outcomes of broad and general use are protocols and instructions for double-helix-resolution imaging of DNA in liquid, see, e.g., a webinar on this (http://www.bruker.com/service/education-training/webinars/afm.html), which helps AFM manufacturer Bruker and its representation in the UK. Technical feedback from our side has helped Bruker to optimise its products before and after market release. Another outcome is the visualisation of membrane lesions and of prepore-state bacterial toxins diffusing on a membrane surface, elucidating the pathways of membrane pore formation by bacterial toxins, as well as understanding of mechanism by which antimicrobial peptides (potential next-generation antibiotics) attack bacteria.
Start Year 2012
 
Description Industrial collaboration with AFM manufacturer JPK Instruments on real-time imaging 
Organisation JPK Instruments
Country Germany 
Sector Private 
PI Contribution We have initiated a collaboration with one of the main commercial manufacturers of atomic force microscopes (AFMs), JPK Instruments in Berlin/Cambridge. JPK has contributed in-kind by AFM controller pre-release hardware and software, and UCL has provided feedback thus enabling JPK to optimise its products.
Collaborator Contribution Technical feedback and suggestions on pre-release AFM equipment.
Impact This has provided JPK Instruments and its represemntation in the UK with means to improve their products.
Start Year 2009
 
Description National Physical Laboratory 
Organisation National Physical Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided the technology to visualise how next-generation antibiotics (de-novo designed antimicrobial peptides) attack bacteria.
Collaborator Contribution Design of antimicrobial peptides. Expertise.
Impact Multidisciplinary, involving physicists, chemists, bioengineers, and biologists. This collaboration has helped to put forward various antimicrobial peptides as (potential) next-generation antibiotics.
Start Year 2010
 
Title A stress-relief layer for functional coatings on micromechanical devices 
Description A disclosure (GB1105100.0) on a method to coat micromechanical devices (e.g., biosensors, cantilevers for atomic force microscopy) with a functional coating without excessively bending them due to coating-induced surface stress. A international (PCT) patent application has been filed. 
IP Reference GB1105100.0 
Protection Patent application published
Year Protection Granted 2011
Licensed No
Impact NA
 
Description Food industry talks 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact At Leatherhead Food Research and at Mars Corporation, talk on nanotechnology sparked questions about the use of it for food industry.

Follow-up emails.
Year(s) Of Engagement Activity 2012,2014
 
Description Lego4Nano workshop in Beijing 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact My PhD students provided the expertise in atomic force microscopy that was essential in this UK-China workshop, sponsored by Lego, to build a low-cost microscope

the workshop was widely reported in the media, including Chinese national newspapers.
Year(s) Of Engagement Activity 2013,2014
URL http://www.london-nano.com/news-and-events/news/2014-lego2nano-launches
 
Description School talks 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Our school talks typically sparked questions and discussions about nanotechnology, about physics and biology.

Follow-up emails.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014