This project is co supervised with Buzz Baum

Fluorescent proteins (FPs) are small (~30 kDa), genetically encodable fluorophores derived from proteins first found in nature — most famously the jellyfish GFP. Because FPs can be used to label and visualise a specific protein of interest via gene-fusion, their use has revolutionised cell biology by enabling average and single protein behaviour to be monitored in living cells and tissues.

Despite being only composed of carbon, nitrogen, oxygen and hydrogen (with the occasional sulphur atom), FPs exhibit a similar brightness and range of colours as more artificial fluorophores. However, their greater sensitivity to light, heat and chemical attack has limited their use for many applications, especially in live imaging in extremophiles and in super-resolution microscopy.

To overcome this problem, we recently began investigating FP photophysics and chemistry to better understand the limits at which they fail to function, using the remarkable behaviour of a recently discovered, naturally-occurring, highly stable fluorescent protein, StayGold, as a standard. Building upon the information gained from these studies, we now seek to further develop our understanding of these complex, light-reactive molecules and use this knowledge to engineer better genetically-encodable fluorophores.

In this project, a PhD student will take this work forward by using directed evolution, rational design, crystallography, fluorescence spectroscopy, transient absorption spectroscopy, and optical triplet depletion to create a new generation of highly stable fluorescent proteins. As well as providing useful probes for the imaging community, this work will directly feed into ongoing projects within the institute on multispectral imaging, cryofluorescence microscopy, imaging flow cytometry, and the live imaging of extremophiles. We therefore expect this work to lead to the development of important biological discoveries.

As a broad and flexible project that can be tailored to the interests of the student, this would suit a physicist, biochemist or physical chemist looking to develop skills in biophysics, protein engineering, and a range of analytical techniques, who is interested in using such approaches to tackle important unsolved fundamental problems in biology.

References

C. Xu, Y. Liu, B.A. Arús, K. Mishra, M.P. Luciano, V.G. Bandi, A. Kumar, Z. Guo, Y. Guan, M.J. Bick, M. Xu, K. Zhang, J. Lingg, J. Bae, A. Kang, S.R. Gerben, A.K. Bera, J.C. Vaughan, J.D. Manton, E. Derivery, M.J. Schnermann, A.C. Stiel, O.T. Bruns, D. Baker (2024)
De novo Design of Near Infrared Fluorescent Proteins
Research Square

A. Kumar, K.E. McNally, Y. Zhang, A. Haslett-Saunders, X. Wang, J. Guillem-Marti, D. Lee, B. Huang, R.R. Kay, D. Baker, E. Derivery, J.D. Manton (2024)
Multispectral live-cell imaging with uncompromised spatiotemporal resolution
bioRxiv

A. Cezanne, B. Hoogenberg, B. Baum (2023)
Probing archaeal cell biology: exploring the use of dyes in the imaging of Sulfolobus cells
Frontiers in Microbiology 14 1233032

B.-J. Chang*, J.D. Manton*, E. Sapoznik, T. Pohlkamp, T.S. Terrones, E.S. Welf, V.S. Murali, P. Roudot, K. Hake, L. Whitehead, A.G. York, K.M. Dean, R. Fiolka (2021)
Real-time multi-angle projection imaging of biological dynamics
Nature Methods 18(7) 829-834

A.A. Pulschen, D.R. Mutavchiev, S. Culley, K.N. Sebastian, J. Roubinet, M. Roubinet, G. Tarrason Risa, M. van Wolferen, C. Roubinet, U. Schmidt, G. Dey, S.-V. Albers, R. Henriques, B. Baum (2020)
Live imaging of a hyperthermophilic archaeon reveals distinct roles for two ESCRT-III homologs in ensuring a robust and symmetric division
Current Biology 30(14) 2852-2859