ProfessorKeith Willison

RESEARCH
Protein folding and CCT

The cytosolic chaperonin CCT is a 1-MDa multi-subunit protein complex that has an essential, core function in actin protein folding in all eukaryotes. In 2011 we determined the atomic structure of the closed form of this yeast CCT-actin complex by X-ray crystallography. The structural studies all point to a remarkably asymmetrical machine with the features required to support a sequential allosteric folding mechanism able to bind specifically and anneal the non-native actin polypeptide. We believe that the final outcome of the CCT-dependent actin folding mechanism is to produce a protein spring whereby the actin monomer is able to reversibly re-explore the folding landscape in the structural context of the polymeric F-actin filament. This model has been extended and refined in my recent review article: Biochemical Journal (2018) 475 3009–3034 - The structure and evolution of eukaryotic chaperonin-containing TCP-1 and its mechanism that folds actin into a protein spring.

You can listen to a 40 minute recorded talk: Willison, K. R. (2021). Chaperonin-containing TCP-1 (CCT), actin springs, and protein folding fluxes. In The Biomedical & Life Sciences Collection, Henry Stewart Talks. Retrieved June 16, 2021, from .

Malarial CCT
Mark Wilkinson, was a 4-year EPSRC Chemical Biology CDT PhD student (2015-2020). Targeting protein folding in the malaria parasite (PhD awarded 2020) with Dr Jake Baum (Life Sciences), Prof Keith Willison (Chemistry).

Truncated latrunculins as actin inhibitors targeting plasmodium falciparum motility and host cell invasion (2016) Journal of Medicinal Chemistry DOI:10.1021/acs.jmedchem.6b01109

Single-molecule nanopore sensing of actin dynamics and drug binding (2020) Chemical science DOI:10.1039/C9SC05710B

A biosynthetic platform for antimalarial drug discovery (2020) Antimicrobial Agents and Chemotherapy DOI:10.1128/AAC.02129-19

SINGLE CELL PROTEOMICS
Single molecule counting approaches are not only an essential approach for clinical and pre-clinical science and systems medicine but also for generating precise quantitative data for the mathematization of biology. The holy grail of single cell proteomics is the ability to count, within high dynamic range, the copy numbers, protein-protein interactions and post-translational modifications of many proteins in individual cells using label-free approaches. Here, label-free means that the proteins are not pre-labelled before analysis: for example by using GFP-gene tagging or in vivo chemical labelling technologies. Prof David Klug and I are working to develop robust, high-throughput methods to count, at single molecule sensitivity, proteins found in bodily fluids and in rare cells present in clinical biopsies. Single molecule detection is highly accurate, does not require calibration and the read-out is digital. We have created a multi-disciplinary laboratory environment to implement and to invent the tools and technologies (T&Ts) needed to achieve these ambitious goals. At this stage in the development of this field most of the T&Ts are by no means plug and play and require expertise in engineering (microfluidics), applied optics (total internal reflection fluorescence and optical trapping spectroscopies), mathematics (data analysis and model building), cell biology (cell separation and manipulation), protein chemistry (antibody development) and chemistry (probe use and development). The first prototype device is the MAC chip (Microfluidic Antibody Capture) which has been used to count p53 tumor suppressor proteins at single molecule level in single colorectal cancer cells. Willison,K.R. and Klug, D.R. (2013) Current Opinion in Biotechnology 24, 745-751.

NANOTECHNOLOGY
We are working with the group of Prof Joshua Edel on various aspects of nanoprobes applied to problems in cell biology and protein dynamics. Our first study on single-cell biopsies was published in Nature Nanotechnology in 2019.

Press release: https://www.imperial.ac.uk/news/189227/nanoscale-tweezers-perform-single-molecule-biopsies-individual/

In 2020 we published a nanopore analysis of actin dynamics and drug binding at single-molecule resolution. We have been able to follow actin unfolding kinetics and F-actin polymerization in a label-free configuration thus opening up fundamentally new approaches to probe the energy landscape of this essential eukaryotic protein.

Single-molecule nanopore sensing of actin dynamics and drug binding


Xiaoyi Wang, Mark D. Wilkinson, Xiaoyan Lin, Ren Ren, Keith R. Willison, Aleksandar P. Ivanov, Jake Baum and Joshua B. Edel


Nanopipettes were used for real-time investigation into actin dynamics and drug binding at single-molecule resolution, showing promise for a better understanding of the mechanism of protein–protein interactions and drug discovery.
From the themed collection:
2019 Chemical Science HOT Article Collection
The article was first published on 03 Dec 2019
Chem. Sci., 2020,11, 970-979
Protein Aggregation
Abeta(40) and α-Synuclein

We have worked with Dr Liming Ying's group on protein aggregation processes concerning the peptides Abeta(40) and α-Synuclein which are involved in human neurodegenerative disease states.
Acetylation Rather than H50Q Mutation Impacts the Kinetics of Cu(II) Binding to α-Synuclein.

Teng X, Sheveleva A, Tuna F, Willison KR, Ying L. Chemphyschem (December 2021)