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Single molecule imaging of transmembrane protein structure and function in their native state

Periodic Reporting for period 3 - TransPhorm (Single molecule imaging of transmembrane protein structure and function in their native state)

Reporting period: 2018-09-01 to 2020-02-29

TransPhorm will pioneer a transformative technology that will enable the study of transmembrane protein (TMP) structure and function in their natural state with unprecedented sensitivity and resolution. TMPs play a crucial role in cell signalling and are essential for life. The focus of this work is on a major class of TMPs called ion channels.

Despite extensive studies, progress in the characterisation of TMPs has been slow and difficult and consequently, no class of proteins has been more difficult to study.

The overarching ambition of TransPhorm is to establish a revolutionary technology to deliver structural, functional and localisation information with unprecedented sensitivity and resolution of TMPs in their native environment. This will deliver tremendous scientific gains and more broadly impact positively on the health and wellbeing of society.

The primary objectives are:
1. Obtain high-resolution structural information of TMPs in their natural state by developing a quantum sensing protocol using Nitrogen Vacancy defects in diamond.
2. Study ion channel function with high temporal resolution and ion selectivity by uniquely employing NV nanodiamonds as magnetic field sensors in solution state nuclear magnetic resonance.
3. Track the location of ion channels in the plasma membrane over time with video rate, multi-modal super resolution optical microscopy.
4. Determine the structure, function and location of ion channels in their natural state through exemplar studies using model TMPs, native TMPs and live cells.
Work to date has concentrated on characterisation and functionalisation of candidate diamond sensors, instrument development for super resolved optically detected magnetic resonance (ODMR) microscopy and development of sensing protocols capable of tracking fast biological process (sub second). Studies applying the developed technology to probe exemplar biological systems represent significant steps towards attainment of the overall aim of project TransPhorm.

An initial focus of the scientific programme was establishment of protocols for integration of diamond sensors with biological systems and diamond functionalisation for targeting specific biological moieties. Work also involved development of strategies to enhance intracellular integration of nanoscale diamond sensors to live cells. For the first time to our knowledge, Nitrogen Vacancy rich nanoscale diamond sensors were integrated into electronspun polymer nanofibers to replicate the physiological niche, particularly important for neuronal and stem cell culture. The electrospun fibre diamond sensing platform supported the growth of neural stem cells and their differentiated progeny and enabled collection of ODMR signals is a physiologically relevant niche. Research studies also demonstrated successful integration of planar diamond chips with sensitive biological systems. The findings of this work provide a path for true integration of ODMR sensing strategies in biologically relevant conditions.

In parallel with development of diamond sensing platforms, instrumentation for optical detection of magnetic resonance and localisation of transmembrane receptors was developed. Indeed, two technical approaches were conceived and deployed to enable fast, super resolution, widefield imaging. Specifically, work produced instrumentation capable of sub-second imaging at double the diffraction limited resolution combining structured illumination microscopy with diamond sensing chips. Further instrumentation development produced a new approach to localisation microscopy based on the intrinsic blinking of NV photoluminescence in a narrow wavelength emission band in physiologically relevant buffers.
This work conceived and demonstrated three novel methodologies to support NV based sensing in biological systems. A previously unreported quantum-sensing platform that mimics the nanoscale architecture and topography of the cell niche has been developed. This sensing platform enabled acquisition of ODMR spectra, longitudinal spin relaxometry measurements and external magnetic field detection. The fibres supported the growth and differentiation of neural stem cells and coordinated firing of neural signals across the cell network. The work advances the current state of the art in quantum sensing by providing a versatile sensing platform that recapitulates physiological-like cell niches and provides fast measurement protocols for detection of co-ordinated endogenous signals from clinically relevant populations of electrically active neuronal circuits.

A further advance is the establishment of a new optical sensing protocol with sub-second time response for monitoring paramagnetic species. The protocol enabled detection of changes in the concentration of paramagnetic ions in proximity to a planar diamond chip containing near surface ensembles of NVs. Experimental work assessed the measurement capabilities of the protocol using solutions of paramagnetic ions (Gd3+, Fe3+), multilamellar liposomes containing Gd3+ labelled phospholipids and a chemical reaction that resulted in the conversion of low spin ferricyanide to high spin hexaaquairon (III). The established protocol operates at room temperature with small sample requirements and a relatively straightforward experimental protocol and data acquisition and analysis. The sub-second time resolution will allow the monitoring of endogenous paramagnetic species production on timescales relevant to discrete signalling pathways related to onset of disease with the scope of aiding therapeutic strategies.

A further advance is the establishment of a novel imaging protocol called self-activated nanodiamond STORM (sandSTORM). This work demonstrated intrinsically-activated photochromism (observed as ‘blinking’) of nanoscale diamonds under low-intensity laser illumination within non-reducing and normoxic media, in a pH- and ionic strength independent manner. Under laser illumination intensities of the order of 100-times lower than that required for conventional localisation microscopy (i.e. dSTORM), NVs blink with an event lifetime that is 5 to 10 times shorter than in dSTORM. Exemplar studies localising transmembrane channel receptors in skeletal muscle cells produced super resolution images 3 to-5 fold faster compared to dSTORM with lateral spatial resolution of the order of 20 nm.

This proposal addresses an important challenge in TMP characterisation that has remained unsolved for over half a century. Results expected by the end of the project will establish a revolutionary technology for structural and functional TMP characterisation with previously unimaginable sensitivity and resolution in their natural environment. TransPhorm will open up new horizons in the understanding of fundamental physiological processes and have tremendous gains in drug discovery especially as ion channels have become the lead target class for many leading novel drugs. The concepts proposed in TransPhorm represent some of the most fertile areas for exploitation of the emerging field of NV based quantum sensing. The strategy and technologies proposed here will take an untraveled path leading to tremendous scientific gains in diverse areas of science and more broadly impact positively on the health and wellbeing of society.