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Mechanical fingerprinting of individual biofibrils using atomic force microscopy

Reference	BB/D011191/1
Principal Investigator / Supervisor	Dr Neil Thomson
Co-Investigators / Co-Supervisors	Professor David Brockwell, Professor Sheena Radford, Professor David Smith
Institution	University of Leeds
Department	Physics and Astronomy
Funding type	Research
Value (£)	276,365
Status	Completed
Type	Research Grant
Start date	02/10/2006
End date	01/10/2009
Duration	36 months

Abstract

This project aims to test the mechanical properties of individual bio-fibril structures, primarily coiled-coils and amyloids. Both can be made from a variety of peptide sources formed through self-assembly in vitro. Coiled-coil fibrils can be engineered from different peptide sources, while amyloids are a common low energy state of a wide variety of peptides and proteins. It will test whether is it possible to obtain characteristic mechanical fingerprints of different fibril types and compare how the mechanical properties of a carefully chosen set of fibrils vary. The fibrillogenic peptides/proteins under study will allow us to examine, the effects of amino-acid sequence, the role of disorder and the effect of higher order structure on the mechanical properties of the resulting fibrils. The systems chosen will give a range of fibril architectures, with stepwise increases in the complexity of fibril structure and morphology allowing a systematic investigation of the various effects. The measurement of the mechanical properties of individual fibrils will be carried out using techniques based on atomic force microscopy. It will yield quantitative data that will allow comparison between fibril types and different architectures. Force spectroscopy, both static and dynamic, will measure their response under axial tension, while nano-scale three point bending tests on nano-structured surfaces will give quantitative numbers of Young's and shear moduli. Once some boundaries of obtainable knowledge have been established and the mechanical response of these varieties characterised, it will then be possible to engineer protein fibril structures with desirable behaviour. Protein fibril structures can be very stable, such as amyloids, or can be activated dynamic systems, e.g. microtubules. Protein fibrils that can be engineered on the nanoscale to have specific mechanical responses and in particular dynamic properties, will enable new soft matter scaffolds and functional devicesto be fabricated. Typically these kinds of fibrils are long (1 to 100 microns or more) and narrow (10 to 50 nm in diameter) and as essentially 1D semi-flexible rods they lend themselves as structural elements and connectors in more complex devices. It is expected that engineered biofibrils will find many applications in bottom-up fabrication of new nanotechonological composite materials and devices. These could include scaffolds for a variety of uses, such as templates for nanowires, meshes for new biocompatible wound sutures, 'rail-roads' in molecular transport devices, biocompatible modifiers of surfaces (e.g. for implant devices) or damping components of new composite materials (e.g. manufactured biominerals).

Summary

Proteins and peptides are linear biological polymer chains that are made up from the 20 different kinds of naturally occurring links (amino acids). Proteins are essentially longer peptide chains, and they fold up into 3D structures, which defines their function. While all proteins are made within the cell, peptides can be artificially synthesised. We have the possibility of using proteins and peptides as functional materials for a variety of uses. These naturally long molecules have the possibility to self-assemble into larger structures, that are often fibril or rod-like in structure. Two major classes of protein self-assembled fibrils are known as coiled-coils and amyloids. These are based on the two major secondary structural elements that peptides and proteins can form when they are folded: alpha-helices and beta-sheets. Coiled-coils occur naturally within structural elements of some proteins in vivo, and are about 2nm in diameter. They are alpha-helices twisted together into a higher order structure: hence the name. They can be engineered in vitro, where many of these coiled-coils assemble head-to-tail lengthways and sideways to form fibrils about 50nm in diameter and microns to millimetres in length. Amyloid fibrils are formed through the self-assembly of beta-sheets into a long continuous structures known as cross-beta where the peptide backbones lie perpendicular to the fibril axis. Several beta-sheets can stack on top of each other to form fibrils between 2 and 5nm across, known as protofilaments. These in turn can twist together into helices and twisted ribbons to form fibrils up to 10 or 20nm in diameter and 10s or 100s of microns in length. In the body, amyloid fibrils are associated with degenerative diseases such as Alzheimer's. The current great challenge for materials engineering in the field of nanotechnology is to be able to control the structure and properties of materials at the smallest possible scales, i.e. the nanometre. This will enable theproduction of objects with unparalleled precision and fidelity and potentially allow us to make and use materials in a more energy efficient manner and to make materials that are themselves functional (i.e. they respond to external stimuli) rather than being static components of a bigger machine or structure. This idea is known generally as bottom-up nano-fabrication. Protein fibrils are interesting materials to engineer for many reasons: they are essentially 1D structures which makes them suitable to use as structural materials or scaffolds; they are biocompatible; they are very stable structures and potentially have a high strength-to-weight ratio; the amino acid sequence space that can be used to engineer different fibril types is vast; and they have the potential to adsorb large amounts of energy efficiently and thereby be useful as a damping component in nano-composite materials. The applications that these materials could have range from scaffolds for molecular wires to artificial wound sutures. This project will use the technique of atomic force microscopy (AFM) to probe the mechanical properties of biological fibrils formed from a variety of peptide and protein sources. The atomic force microscope is an imaging and manipulating device that uses a sharp tip attached to the end of a flexible cantilever spring. The tip can be scanned across surfaces to produce images of molecules while the forces that the tip experiences can be determined by measuring the deflection of cantilever (which is achieved through means of a laser beam reflected from the back of the cantilever). The tip can pick up molecules and measure the forces between different molecules or the strength of molecular structures. The aim of this project to use AFM to investigate how the mechanical properties of different fibril types, made from engineered peptides and full-length proteins, vary and to what extent we can control and modify those properties for use in nano-engineering.

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Technology and Methods Development
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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