High resolution single-molecule observation of functional F1FO complex
Lead Research Organisation:
University of Oxford
Department Name: SABS CDT
Abstract
Background
Many crucial biological processes in cells require energy, and energy transduction is only one of the many important biological processes that are located at cell membranes. The central process in bioenergetics is the synthesis of high-energy ATP molecules using a transmembrane electrochemical gradient called the Protonmotive Force. ATP-synthase F1FO consists of two complementary rotary motors - transmembrane FO and water-soluble F1 - with linked rotors and linked stators. While the rotational mechanism of F1 has been characterized on the single molecule level [1], the requirement for FO to be reconstituted in a sealed lipid bilayer supporting an electrochemical gradient means that it is much less well understood.
The aim of this project will be to achieve high resolution single-molecule observation of functional F1FO complex under controlled membrane voltage and concentration of relevant molecules.
Our Methods
Vesicle fusion [3]
Delivery of membrane proteins into a lipid bilayer is achieved using proteoliposomes with lipids of opposite charge to the target membrane. We recently demonstrated that this fast, one-step delivery method can be used for incorporating large, fragile integral membrane proteins without loss of function, and mixing them with other chosen membrane proteins [3]. Specifically, we mixed F1FO ATP-synthase with a metabolic proton pump in several different lipid bilayer configurations all of which subsequently synthesized ATP.
Droplet on Hydrogel Bilayer (DHB) [4]
Using only a nanoliter water droplet in a lipid-in-oil solution, we can combine single-molecule observation and tracking of integral membrane proteins with voltage clamping of the DHB and current recordings [4].
Vesicle fusion offers for the first time the ability to deliver in principle any membrane protein into a DHB, in contrast to earlier experiments where only the highly robust self-incorporating toxin a-hemolysin was delivered. Unpublished developments in the Berry lab have added the option of perfusion of the liquid environment inside the droplet using a microfluidic device.
An ultrafast protocol for reconstituting F1FO into proteoliposomes [5] and their consequent delivery into Droplet on Hydrogen Bilayers was recently established in the Berry lab. F1FO can be labeled with a gold nanoparticle and its rotation tracked with ultra-high angular and temporal resolution, while concentrations of protons, sodium ions, ATP, ADP and Pi molecules are fully controlled. Moreover, the method will allow us for the first time to directly control the voltage across the bilayer and observe consequent rotation.
This project falls within the EPSRC Biophysics and Soft Matter Physics research area. The project is co-supervised by Dr Mathew Pletcher and Dr Jeffrey Hermes from Roche.
References
[1] Bilyard, T., et al. Philos Trans R Soc Lond B Biol Sci, 2013. 368(1611): p. 20120023.
[2] Gao, Y.Q. et al. Cell, 2005. 123(2): p. 195-205.
[3] Ishmukhametov, R., Nat Commun, 2016. 7, 13025 doi: 10.1038/ncomms13025.
[4] Heron, A.J., et al. J Am Chem Soc, 2009. 131(5): p. 1652-3.
[5] Ishmukhametov, R., et al. Biochim Biophys Acta, 2005. 1706(1-2): p. 110-6.
Many crucial biological processes in cells require energy, and energy transduction is only one of the many important biological processes that are located at cell membranes. The central process in bioenergetics is the synthesis of high-energy ATP molecules using a transmembrane electrochemical gradient called the Protonmotive Force. ATP-synthase F1FO consists of two complementary rotary motors - transmembrane FO and water-soluble F1 - with linked rotors and linked stators. While the rotational mechanism of F1 has been characterized on the single molecule level [1], the requirement for FO to be reconstituted in a sealed lipid bilayer supporting an electrochemical gradient means that it is much less well understood.
The aim of this project will be to achieve high resolution single-molecule observation of functional F1FO complex under controlled membrane voltage and concentration of relevant molecules.
Our Methods
Vesicle fusion [3]
Delivery of membrane proteins into a lipid bilayer is achieved using proteoliposomes with lipids of opposite charge to the target membrane. We recently demonstrated that this fast, one-step delivery method can be used for incorporating large, fragile integral membrane proteins without loss of function, and mixing them with other chosen membrane proteins [3]. Specifically, we mixed F1FO ATP-synthase with a metabolic proton pump in several different lipid bilayer configurations all of which subsequently synthesized ATP.
Droplet on Hydrogel Bilayer (DHB) [4]
Using only a nanoliter water droplet in a lipid-in-oil solution, we can combine single-molecule observation and tracking of integral membrane proteins with voltage clamping of the DHB and current recordings [4].
Vesicle fusion offers for the first time the ability to deliver in principle any membrane protein into a DHB, in contrast to earlier experiments where only the highly robust self-incorporating toxin a-hemolysin was delivered. Unpublished developments in the Berry lab have added the option of perfusion of the liquid environment inside the droplet using a microfluidic device.
An ultrafast protocol for reconstituting F1FO into proteoliposomes [5] and their consequent delivery into Droplet on Hydrogen Bilayers was recently established in the Berry lab. F1FO can be labeled with a gold nanoparticle and its rotation tracked with ultra-high angular and temporal resolution, while concentrations of protons, sodium ions, ATP, ADP and Pi molecules are fully controlled. Moreover, the method will allow us for the first time to directly control the voltage across the bilayer and observe consequent rotation.
This project falls within the EPSRC Biophysics and Soft Matter Physics research area. The project is co-supervised by Dr Mathew Pletcher and Dr Jeffrey Hermes from Roche.
References
[1] Bilyard, T., et al. Philos Trans R Soc Lond B Biol Sci, 2013. 368(1611): p. 20120023.
[2] Gao, Y.Q. et al. Cell, 2005. 123(2): p. 195-205.
[3] Ishmukhametov, R., Nat Commun, 2016. 7, 13025 doi: 10.1038/ncomms13025.
[4] Heron, A.J., et al. J Am Chem Soc, 2009. 131(5): p. 1652-3.
[5] Ishmukhametov, R., et al. Biochim Biophys Acta, 2005. 1706(1-2): p. 110-6.
Planned Impact
The main impact of the SABS CDT will be the difference made by the scientists trained within it, both during their DPhils and throughout their future careers.
The impact of the students during their DPhil should be measured by the culture change that the centre engenders in graduate training, in working at the interface between mathematical/physical sciences and the biomedical sciences, and in cross sector industry/academia working practices.
Current SABS projects are already changing the mechanisms of industry academic collaboration, for example as described by one of our Industrial Partners
"UCB and Roche are currently supervising a joint DPhil project and have put in two more joint proposals, which would have not been possible without the connections and the operational freedom offered by SABS-IDC and its open innovation culture, a one-of-the-kind in UK's CDTs."
New collaborations are also being generated: over 25% of current research projects are entirely new partnerships brokered by the Centre. The renewal of SABS will allow it to continue to strengthen and broaden this effect, building new bridges and starting new collaborations, and changing the culture of academic industrial partnerships. It will also continue to ensure that all of its research is made publically available through its Open Innovation structure, and help to create other centres with similar aims.
For all of our partners however, the students themselves are considered to be the ultimate output: as one our partners describes it,
"I believe the current SABS-IDC has met our original goals of developing young research scientists in a multidisciplinary environment with direct industrial experience and application. As a result, the graduating students have training and research experience that is directly applicable to the needs of modern lifescience R&D, in areas such as pharmaceuticals and biotechnology."
However, it is not only within the industrial realm that students have impact; in the later years of their DPhils, over 40% of SABS students, facilitated by the Centre, have undertaken various forms of public engagement. This includes visiting schools, working alongside Zooniverse to develop citizen science projects, and to produce educational resources in the area of crystal images. In the new Centre all students will be required to undertake outreach activities in order to increase engagement with the public.
The impact of the students after they have finished should be measured by how they carry on this novel approach to research, be it in the sector or outside it. As our industrial letters of support make clear, though no SABS students have yet completed their DPhils, there is a clear expectation that they will play a significant role in shaping the UK economy in the future. For example, as one of our partners comments about our students
"UCB has been in constant search for such talents, who would thrive in pharmaceutical research, but they are rare to find in conventional postgraduate programmes. Personally I am interested in recruiting SABS-IDC students to my group once they are ready for the job market."
To demonstrate the type of impact that SABS alumni will have, we consider the impact being made by the alumni of the i-DTC programmes from which this proposal has grown. Examples include two start-up companies, both of which already have investment in the millions. Several students also now hold senior positions in industry and in research facilities and institutes. They have also been named on 30 granted or pending patents, 15 of these arising directly from their DPhil work.
The examples of past success given above indicate the types of impact we expect the graduates from SABS to achieve, and offer clear evidence that SABS students will become future research leaders, driving innovation and changing research culture.
The impact of the students during their DPhil should be measured by the culture change that the centre engenders in graduate training, in working at the interface between mathematical/physical sciences and the biomedical sciences, and in cross sector industry/academia working practices.
Current SABS projects are already changing the mechanisms of industry academic collaboration, for example as described by one of our Industrial Partners
"UCB and Roche are currently supervising a joint DPhil project and have put in two more joint proposals, which would have not been possible without the connections and the operational freedom offered by SABS-IDC and its open innovation culture, a one-of-the-kind in UK's CDTs."
New collaborations are also being generated: over 25% of current research projects are entirely new partnerships brokered by the Centre. The renewal of SABS will allow it to continue to strengthen and broaden this effect, building new bridges and starting new collaborations, and changing the culture of academic industrial partnerships. It will also continue to ensure that all of its research is made publically available through its Open Innovation structure, and help to create other centres with similar aims.
For all of our partners however, the students themselves are considered to be the ultimate output: as one our partners describes it,
"I believe the current SABS-IDC has met our original goals of developing young research scientists in a multidisciplinary environment with direct industrial experience and application. As a result, the graduating students have training and research experience that is directly applicable to the needs of modern lifescience R&D, in areas such as pharmaceuticals and biotechnology."
However, it is not only within the industrial realm that students have impact; in the later years of their DPhils, over 40% of SABS students, facilitated by the Centre, have undertaken various forms of public engagement. This includes visiting schools, working alongside Zooniverse to develop citizen science projects, and to produce educational resources in the area of crystal images. In the new Centre all students will be required to undertake outreach activities in order to increase engagement with the public.
The impact of the students after they have finished should be measured by how they carry on this novel approach to research, be it in the sector or outside it. As our industrial letters of support make clear, though no SABS students have yet completed their DPhils, there is a clear expectation that they will play a significant role in shaping the UK economy in the future. For example, as one of our partners comments about our students
"UCB has been in constant search for such talents, who would thrive in pharmaceutical research, but they are rare to find in conventional postgraduate programmes. Personally I am interested in recruiting SABS-IDC students to my group once they are ready for the job market."
To demonstrate the type of impact that SABS alumni will have, we consider the impact being made by the alumni of the i-DTC programmes from which this proposal has grown. Examples include two start-up companies, both of which already have investment in the millions. Several students also now hold senior positions in industry and in research facilities and institutes. They have also been named on 30 granted or pending patents, 15 of these arising directly from their DPhil work.
The examples of past success given above indicate the types of impact we expect the graduates from SABS to achieve, and offer clear evidence that SABS students will become future research leaders, driving innovation and changing research culture.
People |
ORCID iD |
| Richard Berry (Primary Supervisor) | |
| Zuzana Coculova (Student) |