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Quantification of the Dynamic of Protein Complexes


Project Description

In many diseases, including infection and inflammation, the cellular response to extracellular stimuli (e.g. pathogens) is regulated by membrane receptor signalling representing more than 50% of today’s clinical drug targets. Failure and negative side effects of many of the current drugs are related to a lack in tools for investigating the various mechanisms of receptor activation and protein-protein interactions. Open questions are related to receptor clustering, endosomal signalling, transmission thresholds, and pathogen interference impacting signal strength and specificity. [1]

My lab has been working on different approaches for counting protein copy numbers in cellular structures including my own method, “counting on photon statistics” (CoPS). We have developed protocols to reliably relate the number of fluorescent labels to the actual protein copy numbers in living cells and use these methods to quantify T cell receptor activation and study its modulation by the HIV protein Nef. [2]

In this context, we seek to establish robust methods for counting copy numbers and measuring the dynamics of proteins participating in T-cell signalling using single-molecule fluorescence spectroscopy. [3] Furthermore, we plan to transform the existing confocal technique to an imaging approach to enable protein quantification in cellular structures and eventually in whole cells.

The project aims at investigating existing single-molecule approaches, like Counting on Photon Statistics (CoPS) and single-molecule intensity analysis, for their use to quantitatively measure association/dissociation dynamics of protein assemblies.

Project outline:

Year 1: Preparation of standardized samples and setup of the single-photon sensitive confocal microscope

DNA-origami with a defined number of single-stranded binding sites for fluorescently labelled complementary DNA-oligonucleotides will be used to compare existing methods in vitro. The kinetic range of hybridisation/denaturation will be modelled by systematic variation of the complementary DNA-oligonucleotide length. Reversible hybridization kinetics will be characterized by single-molecule TIRF-experiments and subsequent analysis of the resulting intensity transients for a different number of binding sites (e.g. 1, 4, 10, etc. binding sites). The design of the DNA-origami may be carried out in collaboration with Philip Tinnefeld (LMU Munich). The binding kinetics will be measured for a single binding site in comparison to multiple binding sites. [4] Key questions concern the experimental limits of kinetic measurements:

- What is the maximal number of binding sites allowing kinetic measurements?
- What are the respective concentration ranges?
- What dynamic range can be achieved, i.e. fastest vs. slowest binding kinetics?

In the first year, the student will start learning the preparation of DNA-origami samples and single-molecule TIRF/wide-field microscopy. Samples can be prepared in the Institute of Cardiovascular Sciences or School of Chemistry with support from Robert Neely, while single-molecule experiments will be done in the microscopy facility of the Institute of Cardiovascular Sciences. Ideally, the student will be involved in setting up the microscope in spring 2020 to lear the experimental technique (CoPS) and the data analysis. [3] Two postdocs from Heidelberg will also join my lab in Birmingham to setup labs and microscopes and to support the PhD student in this project.

Year 2: Comparison of the single-molecule TIRF approach with CoPS

Year 1 results will be complemented with experiments on a single-photon sensitive confocal microscope with CoPS measurements. The number of bound DNA-oligonucleotides for each time point will be estimated from the recorded time-resolved photon statistics using established analysis algorithms. It was shown that the counting range of CoPS exceeds the range of alternative approaches, e.g. single-step photo bleaching. [2, 3] Therefore, the same key questions will be addressed for dynamic measurements to learn if and to which extent kinetic measurements can be improved by CoPS. The work will be summarized and presented on national and international conferences as well as published as a benchmark of single-molecule kinetics and dynamic CoPS measurements. The results will allow to determine suitable kinetic and concentration ranges and enable identification of applicable biological targets for studies in year 3.

Year 3: Extension of the kinetic measurements to relevant biological targets

The studies will be extended to protein complexes, e.g. artificial Thrombin complexes on DNA-aptamers, [5] and receptor clusters, e.g. GPCR clusters on nanodiscs (Steve Hill, COMPARE). [6] The successful method will also be used to study the dynamics of membrane protein complexes in live cell experiments. Different existing constructs of adaptor proteins (LAT-Halo and SLP76-Halo) involved in TCR micro cluster formation will be used to study micro cluster formation and reorganization during T-cell activation. [7-9] The measurement may be complemented with the recently established serial sectioning approach to enable a quantitative 3D reconstruction of cellular structures.

Funding Notes

This is a 3-year PhD fellowship funded by COMPARE (The Centre of Membrane Proteins and Receptors). COMPARE seminars offer opportunities to learn about potential collaborations in the field of membrane protein/receptor clustering and to present the progress of the PhD project on a regular basis.

The project is linked to a second COMPARE PhD studentship dealing with improving data analysis in quantitative microscopy co-supervised by Iain Styles (School of Computer Sciences) which should support the experimental work in project.

Interested students should contact Prof Herten (), with a cover letter and CV.

References

[1] Meng et al. Nat. Rev. Cardiol. 13, 167 (2016), doi: 10.1038/nrcardio.2015.169.
[2] Grussmayer et al. Meth. Appl. Fluoresc. 7, 012003 (2019), doi: 10.1088/2050-6120/aaf2eb.
[3] Grussmayer et al. PCCP 19, 8962 (2017), doi: 10.1039/C7CP00363C.
[4] Jungmann et al. Nano Lett. 10, 4756 (2010), doi: 10.1021/nl103427.
[5] Chhabra et al. JACS 129, 10304 (2007), doi: 10.1021/ja072410u.
[6] Jaeger et al. Front. Endocrinol. 5, 26 (2014), doi: 10.3389/fendo.2014.00026.
[7] Abraham et al. J. Immunol. 189, 1898 (2012), doi: 10.4049/jimmunol.1200652.
[8] Pan et al. Cell Microbiol. 15, 1605 (2013), doi: 10.1111/cmi.12148.
[9] Abraham et al. Cell Commun. Signal. 10, 39 (2012), doi: 10.1186/1478-811X-10-39.


More information:

The Centre of Membrane Proteins and Receptors (COMPARE): http://www.birmingham-nottingham.ac.uk/compare/

The Institute of Cardiovascular Sciences: https://www.birmingham.ac.uk/research/activity/cardiovascular-sciences/index.aspx

The School of Chemistry: https://www.birmingham.ac.uk/schools/chemistry/index.aspx

Postgraduate studies at the University of Birmingham: https://www.birmingham.ac.uk/postgraduate/prospectus/index.aspx

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