Award details

Mapping the complexity of protein unfolding landscapes using novel mechanical unfolding experiments

Reference	BB/C507029/1
Principal Investigator / Supervisor	Professor Sheena Radford
Co-Investigators / Co-Supervisors	Professor David Brockwell
Institution	University of Leeds
Department	Physics and Astronomy
Funding type	Research
Value (£)	258,328
Status	Completed
Type	Research Grant
Start date	31/01/2005
End date	30/01/2008
Duration	36 months

Abstract

Conventional measurements of protein folding and unfolding obscure important details of the complex energy landscape by the very nature of these averaged measurements. By contrast, single molecule techniques, in principle, permit individual folding pathways to be observed. One powerful single molecule method is mechanical manipulation using the atomic force microscope (AFM). A unique advantage of this technique is that force can be applied to a protein in different directions, allowing the unfolding reaction coordinate to be clearly defined. Moreover, the technique can be used to probe the local stability of structural elements, rather then the global stability measured by most other techniques, allowing new information about structure stability and cooperativity of different protein structures to be obtained. In this project we will explore the unfolding landscape of three different proteins (I27, E2lip3 and protein L) by a series of experiments in which either the sequences of the protein is altered and/or and same protein is unfolded by applying force in different directions. This can be achieved by a novel strategy that permits attachment of the protein to the substrate at specific points. Using these experiments we will determine (i) the effect of pulling geometry on the mechanical unfolding properties of I27; (ii) detailed information about the structure of the unfolding transition state of E2lip3 when the protein is pulled in two different directions and (iii) the fundamental origins of mechanical resistance by mutagenesis experiments and solvent perturbation experiments on a small, simple protein (protein L) that we have recently shown to be an ideal model system for forced unfolding studies. These experiments will also allow us to test predictions of the effect of pulling geometry and amino acid sequence on the mechanical stability of these three proteins obtained using steered molecular dynamics simulations. The outcomes will be new insights into the origins of mechanical resistance of these protein domains and a robust benchmarking of the outputs of molecular dynamics simulations of mechanical unfolding which will guide the field in future developments in both experiment and simulation.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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