BBCSRC David Phillips Fellowship Manipulating biomolecule conformational dynamics and biochemical reactions at single molecule level

Lead Research Organisation: Imperial College London
Department Name: National Heart and Lung Institute

Abstract

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Technical Summary

Our objective is to develop the next generation single molecule tools by integrating single molecule spectroscopic methodologies with the controlled delivery of biomolecules from a nanopipette and apply them to important biological problems that have been intractable to date. This can be used to answer key questions such as the number of microscopic pathways in a folding reaction and investigate the existence of the stochastic resonance and memory effect in enzymatic turnover.

Publications

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Clarke RW (2007) Surface conductivity of biological macromolecules measured by nanopipette dielectrophoresis. in Physical review letters

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Fegan A (2010) Ensemble and single molecule FRET analysis of the structure and unfolding kinetics of the c-kit promoter quadruplexes. in Chemical communications (Cambridge, England)

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Gilburt J (2017) Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy in Angewandte Chemie

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Gilburt JAH (2017) Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy. in Angewandte Chemie (International ed. in English)

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Huang F (2007) Distinguishing between cooperative and unimodal downhill protein folding. in Proceedings of the National Academy of Sciences of the United States of America

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Huang F (2009) Direct observation of barrier-limited folding of BBL by single-molecule fluorescence resonance energy transfer. in Proceedings of the National Academy of Sciences of the United States of America

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Huang F (2009) Multiple conformations of full-length p53 detected with single-molecule fluorescence resonance energy transfer. in Proceedings of the National Academy of Sciences of the United States of America

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Mehta P (2013) Dynamics and stoichiometry of a regulated enhancer-binding protein in live Escherichia coli cells. in Nature communications

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Piper J (2008) Characterization and Application of Controllable Local Chemical Changes Produced by Reagent Delivery from a Nanopipet in Journal of the American Chemical Society

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Piper JD (2006) A renewable nanosensor based on a glass nanopipette. in Journal of the American Chemical Society

 
Description There is a challenge to develop new quantitative physical tools to interrogate the complexity of biology on a molecular basis. These tools require the properties of high specificity, nanometre spatial resolution, millisecond time resolution and single molecule sensitivity. The main objective of the fellowship is to develop novel single molecule tools by integrating single molecule spectroscopic methodologies with the controlled delivery of biomolecules from a ~ 100 nanometre glass pipette and apply them to address biological problems that have been difficult to investigate using conventional methods.

Two single molecule fluorescence microscopes have been constructed during the fellowship, one for in vitro single molecule fluorescence studies and the other for in vivo living cell imaging. These instruments have been adapted to become versatile nano-tools which are capable of functional imaging of the living cell with high spatial resolution, controlled delivery and nanowriting of biomolecules on functionalised glass surface, concentrating and trapping of biomolecules. They were also used for colour switching of dual-labelled DNA, renewable nanosensing of metal ions and pH, as a nanomixer for single molecule kinetics measurement and for single molecule triggering and single cell manipulation. These new methodologies have opened new windows for future advanced single molecule studies in living cells combing both the single molecule imaging and single molecule manipulation.

Single molecule fluorescence resonance energy transfer (smFRET) has been used to investigate intramolecular DNA quadruplex formed in the human telomeric repeat sequence and in the promoter regions of several human genes. Conformational heterogeneity of these quadruplexes has been proved to be ubiquitous and the role of DNA quadruplex in the regulation of gene expression has been envisaged. smFRET has also been applied to study the folding mechanism of small fast folding proteins such as B-domain protein A and E3BD. The results support a two-state folding scenario, suggesting that no protein folds without a free energy barrier under chemical denaturant conditions. smFRET studies on the tumour suppressor p53, a guardian of the human genome, revealed multiple conformations of full-length p53, which remain unnoticed by conventional techniques.
Exploitation Route The controlled delivery via a glass nanopipette technology the PI co-developed has been patented in the US, EU and internationally and is one of the selling points of the product of the start-up company Ionscope Ltd which is commercialising scanning ion conductance microscope.
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description The two single molecule fluorescence imaging microscopes constructed has been further used for live cell imaging and several PhD student projects. The nanopipette delivery technique has been used and improved by several other groups.
First Year Of Impact 2007
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic