Intracellular Biophotonic Nanoswitches

Lead Research Organisation: Cardiff University


Recent mapping of all physical interactions between proteins in a given cell has confirmed the notion that interactions between proteins are highly regulated and underpin all cellular processes. Researchers and technologists have been presented with a major challenge - how to ask specific questions of such complex systems especially when protein interactions change with time in a given cell and result in different end states. For example when a human cell responds to stress, specific interactions between master regulatory proteins start to drive a recovery process or initiate a controlled commitment to cell death. This project aims to generate a generic technology for solving this problem - introducing synthetic switches into live cells that can 'fine-tune' protein interactions by remote control. This exciting approach, based on highly promising preliminary work, would allow the investigator to programme changes in defined protein-protein interactions by the introduction of small interfering molecules engineered to be switched on and off by light of carefully selected wavelengths. Changes in the structure of a small molecule are triggered by external light pulses inducing conformational rearrangements in the peptide backbone and hence alterations of the biological properties of the Intracellular Biophotonic Nanoswitch (IBN). IBNs are light-sensitive nanoparticle-based molecular structures linked to the short peptide sequences that recognize features on the surface of a protein that has been targeted for switching. Conventional and novel methods for IBN delivery into live cells will allow patterning of the swiches into populations of cells. Operating these IBNs by light will allow the researcher to pattern the activation of switches in such complex cell populations or to 'programme' the switching process in single cells - a step-forward in the technology of manipulating master regulators of discrete intracellular pathways. Our proposal's adventure and risk relates to the problems of IBN design and their potential for self-reporting in live cells.IBNs will allow a researcher to switch or programme the state of a master regulator in a live cell by biophysical means and explore the consequences on the whole system to reveal the internal linking of different pathways. Furthermore, our proposal addresses how to track the downstream consequences of selective switching, even in different lineages, to reveal how cells respond to different signals (amplitude or frequency) in developing their responses even if these arise quickly or indeed develop slowly through different cell generations.Since our vision is to provide the life sciences community with novel, robust and readily implemented technologies based on robust chemical systems, the proposal encompasses engagement with potential user & downstream demands of IBN technology with a focus on the burgeoning demand to understand cellular biology at the complex 'systems' level. The exciting prospect looms of gaining programmable photonic control over normal physiology (directing stem cell differentiation, manipulating wound healing and delaying cell senescence), neoplasia (cancer biology of cell cycle checkpoint dysfunction and photonically-controlled therapeutics), constructed cell communities (light-directed tissue engineering) and molecular target identification (the search for new medicines and products).


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Wysoczanski P (2012) NMR solution structure of a photoswitchable apoptosis activating Bak peptide bound to Bcl-xL. in Journal of the American Chemical Society

Description Selective protein-protein interactions underpin the processes of life and the construction of equally selective tools to examine protein-protein interactions underpins our attempts to understand cellular behaviour at a molecular level. Among the most important networks are those that allow a cell to commit to programmed cell death and remove themselves from the body if their genetic material becomes dangerously damaged. Our approach investigated this network by taking small peptide fragments from larger proteins and constraining their structure with a dye molecule. This dye molecule changes shape when irradiated with a particular wavelength of light and was used to control the conformation of peptides to create photo-responsive inhibitors of protein-protein interactions. Theoretically, this technique is perfect for signalling networks where the frequency rather than the magnitude of a molecular signal carries information.

The structure of the complex between our azobenzene peptide and its natural target protein Bcl-xL was solved by nuclear magnetic resonance (NMR) spectroscopy. This involves recording a number of two and three dimensional correlation spectra that allow the assignment of NMR peaks for the individual amino acid residues in the protein and peptide. The spectra can them be used to estimate the distance between residues to build up a list of structural restraints and hundreds of these restraints are used to calculate the most likely structure. The final calculated structure showed only minor changes to the structure of the complex compared to the structure obtained with an unmodified peptide. The azobenzene complex closely resembled other known proapoptotic helices bound to Bcl-xL, suggesting the introduction had not unduly changed the binding mode of the peptide.

Photoswitchable peptides were next introduced to cells. The problem of variable uptake across a cell population proved a major challenge, but was addressed by the careful use of a natural product, digitonin, that forms pores in the outer membrane of cells that are large enough to allow uniform entry of our azobenzene peptides to the cytosol of a population of cells but are small enough to retain the proteins constituting the intrinsic apoptosis pathway. The effect of our peptides could be monitored by observing a fluorescent dye, JC-1, which changes colour from red to green when a cell where mitochondria are depolarised: an early sign of apoptosis. By employing fluorescence activated cell sorting analysis we can determine the mitochondrial health of a large number of independent cells and gauge the effect of our peptides.
Exploitation Route The paper describing our NMR structure has been cited numerous times by researchers interested in design of drugs targeting protein-protein interactions.

With our assistance, our collaborators in the School of Medicine are testing the potential of our peptides to profile cell responses in order to predict the most appropriate therapeutic approaches on a patient-by-patient basis.
Sectors Healthcare

Description Articles describing work from this grant has appeared on the BBC News website and a number of science and technology news sites, engaging interested members of the public who constructed a Wikipedia page dedicated to photoactivated peptides.
Title Cell Profiling Peptides 
Description Further funding was received in a bid with Prof. Rachel Errington of the Cardiff University School of Medicine from the MRC Proximity to Discovery - Industrial Engagement Fund to develop our conformationally restricted peptides into predictive diagnostic tools in collaboration with a major phormaceutical company. 
Type Diagnostic Tool - Non-Imaging
Current Stage Of Development Initial development
Year Development Stage Completed 2016
Development Status Actively seeking support
Impact At this early stage of development none have arisen yet. 
Description Web News Coverage (Flash of Light) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Article on Science and Environment section of BBC News website.

No direct contact with readers to judge effect.
Year(s) Of Engagement Activity 2010