Spatial dynamics of electron transport

Lead Research Organisation: Queen Mary, University of London
Department Name: Sch of Biological and Chemical Sciences

Abstract

Life depends on processes of energy conversion, in which energy obtained from sunlight, or from catalysing chemical reactions such as the breakdown of food molecules, is converted into energy in forms that can be used to power the biochemistry of the cell. Some key biological energy conversion processes take place in membranes and involves the transport of electrons from donors to acceptors, powered by either light (photosynthesis) or chemical energy (respiration). These processes require a set of membrane components including protein complexes and smaller molecules that can transport electrons in the membrane. The components involved are now well understood, but their organisation and interactions in the intact membrane are much less understood. In some cases there are alternative possible routes for electron transport. Electron transport routes are regulated by physiological factors, and have strong effects on the physiology of the cell. It seems clear that the pathways of electron transport must depend somehow on the organisation of the electron transport components in the membrane. However, it remains unclear on what length scales electron transport takes place (i.e. how far in the membrane do electrons travel from their initial donor to their final acceptor) and on what length scales complexes might be organised in order to control electron transport pathways. Knowing the answers to these questions could give us powerful new tools for controlling biological energy conversion, allowing the engineering or organisms suitable for more efficient biofuel production, for example. We are using a cyanobacterium (a kind of photosynthetic bacterium) as a model organism. In cyanobacteria both photosynthesis and respiration occur in a complex membrane system inside the cell called the thylakoid membranes. Our initial approach to understanding the control of electron transport routes in cyanobacteria has used techniques in which we generate mutants in which particular electron transport proteins are fused to a fluorescent protein. We can then use a fluorescence microscope to see the distribution of electron transport proteins in intact membranes in living cells. A disadvantage of fluorescence microscopy is that it has relatively low resolution, i.e. we cannot see the organisation of electron transport components at molecular scales. However, the technique has allowed us to observe that the distribution of electron transport complexes is very heterogeneous on the scales that we can observe. Under some conditions particular electron transport components are concentrated into distinct patches in the membrane. We can show that the distribution of complexes into patches, or otherwise, is under physiological control, and we can relate this to the physiological control of electron transport. Our aims in this project are to understand better how the membrane patches are formed, what they contain, how they are organised and what effects they have on electron transport. Initially we will use an extension of our current fluorescent labelling techniques to get a more complete picture of the composition of the patches in living cells. This will be combined with studies to test hypotheses for the ways in which the distribution of complexes in the membrane are under physiological control. We will then use biochemical techniques to isolate the membrane patches and determine their full composition, combined with the use of electron microscopy to get higher-resolution information on the organisation of protein complexes in the membrane. At the end of the study we expect to understand much better how electron transport in the membrane is controlled. We expect our studies on a cyanobacterium to act as an exemplar for studies of biological electron transport at the membrane scale in other organisms, and to provide new ideas for the control of the organisation and function of biological membranes in general.

Technical Summary

The thylakoid membranes of cyanobacteria contain both photosynthetic and respiratory electron transport complexes. This allows the possibility of multiple electron transport routes, including routes in which electrons are transferred from respiratory donors to photosynthetic acceptors, and vice versa. The routes taken by electrons are crucial for cellular physiology, since they control both the redox balance of the cell and its main means of energy conversion. The question we wish to address in this proposal is what controls the probability of the different possible electron pathways. The problem in cyanobacteria is a specific example of a more general problem in bioenergetic membranes. Our current results strongly suggest that routes of electron transport in the cyanobacterium Synechococcus 7942 (and in other organisms) are controlled by lateral heterogeneity in the membrane on the sub-micron scale. This heterogeneity is on a large enough scale to be visualisable by GFP-tagging and fluorescence microscopy. In this project we will further define the composition and structure of zones in the Synechococcus membrane in which respiratory complexes are concentrated, initially by further fluorescent tagging and confocal microscopy, then using biochemical approaches and electron microscopy. We will use spectroscopic techniques to determine the effects that membrane heterogeneity has on electron transport pathways. We will investigate the signal transduction mechanisms that regulate the lateral distribution of complexes, in terms of the initial triggering signals and in terms of the downstream factors that lead to re-organisation of the membrane. We will combine this with an investigation of the dynamic behaviour of quinone electron carriers in bioenergetic membranes, in order to get a complete picture of the way in which electron transport pathways in an intact bioenergetic membrane relate to the mobility and distribution of the electron carriers.

Planned Impact

Who will benefit from this research?

a. The wider academic and commercial microbiology community (beyond the investigators' immediate circle in the photosynthesis and bioenergetics research communities). We foresee particular benefits for those wishing to engineer microorganisms for more efficient biofuel production.

b. Academic and industrial researchers who wish to use freeze-fracture electron microscopy as a tool for investigating biological membrane organisation.

How will they benefit from this research?

a. Our research will clarify the role of membrane organisation in controlling electron transport pathways in a bacterial membrane, and in addition we expect to uncover factors controlling the larger scale-distribution of protein complexes in the membrane. We envisage considerable potential benefits of this knowledge for anyone wishing to engineer microorganisms for biofuel production, or any other membrane-associated biochemical pathway.

b. Added value from our project will come from consolidation of a freeze-fracture electron microscopy preparation facility at QMUL, providing a resource that will be open to outside users. FFEM resources are now extremely scarce, both in the UK and world-wide. Yet we believe the technique is due for a renaissance, in part because our research will act as an exemplar of what can be achieved by FFEM in combination with modern image-analysis techniques.

Publications

10 25 50
 
Description 1. We used single-molecule fluorescence techniques to demonstrate that electron transport in the plasma membrane of Escherichia coli is fully delocalised, in contrast to the increasingly prevailing dogma that electron transport occurs within supercomplexes.
2. We developed a method for isolation of respiratory complex 1 (NDH-1) from the thylakoid membranes of a cyanobacterium by affinity pull-downs, and used this method to provide initial evidence for strong association of NDH-1 with Photosystem I - a first clue to the hard-wiring of electron transport pathways in this bacterium.
3. In collaboration, we helped to demonstrate a crucial role for hydrocarbons in shaping the thylakoid membranes of cyanobacteria.
4. We uncovered the basis for directional light-perception in a cyanobacterium, showing that a cyanobacterium can detect (and move towards) light sources because its cells act as spherical lenses that focus an image of the light-source on the opposite edge of the cell.
Exploitation Route All of our key findings open up new avenues of academic enquiry that will be taken forward by ourselves and others. Our demonstration of micro-optical effects in cyanobacterial cells has potentially huge implications for the efficiency of photosynthesis, especially in photosynthetic biofilms. We are currently following up this work, and we believe that it will eventually lead to improved designs for photosynthetic bioreactors for solar-powered biotechnology.
Sectors Energy,Manufacturing, including Industrial Biotechology

 
Description An impact objective was to develop a facility for freeze-fracture electron microscopy (a useful technique where expertise and equipment is becoming increasingly scarce) that is open to academic and industrial users. The facility is now fully established with a facility manager in place.
First Year Of Impact 2014
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description ALERT2017
Amount £302,000 (GBP)
Funding ID BB/R000514/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start  
 
Description Marie Sklodowska-Curie Innovative Training Network
Amount £395,000 (GBP)
Funding ID SE2B 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 03/2016 
End 02/2020
 
Description Research grant
Amount £24,144 (GBP)
Funding ID BB/R003890/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 03/2021
 
Description Atlantic Magazine 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interviewed for article in The Atlantic magazine on our work on "vision" in bacteria.
Year(s) Of Engagement Activity 2016
URL http://www.theatlantic.com/science/archive/2016/02/this-bacterium-acts-like-a-one-cell-eyeball/46050...
 
Description BBC Radio 5 Live 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Live interview on BBC Radio 5 Live breakfast programme, 9/2/2016
Year(s) Of Engagement Activity 2016
URL http://www.bbc.co.uk/news/science-environment-35502310
 
Description BBC World Service 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview on BBC World Service, 9/2/2016
Year(s) Of Engagement Activity 2016
URL http://www.bbc.co.uk/news/science-environment-35502310
 
Description Bacteriofiles podcast 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact "Bacteriofiles" podcast on our work on "vision" in bacteria
Year(s) Of Engagement Activity 2016
URL http://www.bacteriofiles.com/2016/09/bacteriofiles-268-sophisticated.html
 
Description Radio 4 Today Programme 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Live Interview on BBC Radio 4 Today Programme, 9/2/2016
Year(s) Of Engagement Activity 2016
URL http://www.bbc.co.uk/news/science-environment-35502310
 
Description Scientific American interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview with Scientiifc American magazine, in connection with our work on "vision" in bacteria.
Year(s) Of Engagement Activity 2017
URL https://www.scientificamerican.com/article/veggies-with-vision-do-plants-see-the-world-around-them/
 
Description Yale Scientific Magazine 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Interviewed for article in Yale Scientific Magazine on our work on "vision" in bacteria
Year(s) Of Engagement Activity 2016
URL http://www.yalescientific.org/2016/08/follow-the-light-bacteria-in-motion/