BBSRC ALERT 24 - Concurrent force sensing and fluorescence imaging – Enabling the next generation of single molecule biophysics research across Faculties at Leeds

Project title

BBSRC ALERT 24 - Concurrent force sensing and fluorescence imaging – Enabling the next generation of single molecule biophysics research across Faculties at Leeds

Description

Project Overview

Life relies on biomacromolecules – proteins, nucleic acids, carbohydrates and lipids – and their ability to interact with each other and with smaller binding partners in defined ways to form biomolecular complexes. Function typically emerges from biomolecular complexes dynamically changing their structure in response to the physical and chemical stimuli of their environment. Understanding life in molecular detail thus critically relies on our ability to quantify the changes in molecular conformation and interaction rates, over time and as a function of mechanical force, pH or temperature, or the presence of distinct binding partners.Life relies on biomacromolecules – proteins, nucleic acids, carbohydrates and lipids – and their ability to interact with each other and with smaller binding partners in defined ways to form biomolecular complexes. Function typically emerges from biomolecular complexes dynamically changing their structure in response to the physical and chemical stimuli of their environment. Understanding life in molecular detail thus critically relies on our ability to quantify the changes in molecular conformation and interaction rates, over time and as a function of mechanical force, pH or temperature, or the presence of distinct binding partners.
This insight can be difficult to obtain by traditional ‘ensemble’ methods, as these average over the conformational heterogeneity of biomolecular complexes in time and/or space. Complexity is removed by observing biomolecular complexes one at a time using so-called ‘single-molecule’ methods. Over the last 30 years, it has become possible to capture, manipulate and study biomolecular complexes using both force-based methods (such as atomic force microscopy (AFM) and optical tweezers) and fluorescence-based methods. The University of Leeds has a strong track record in single-molecule force (using AFM) and single-molecule fluorescence methods, and also in the interpretation of such experimental data through the lens of theoretical (soft matter physics) models. Optical tweezers can resolve lower forces than AFM, thus providing access to the single piconewton and sub-piconewton regimes that are relevant for many biological processes. Furthermore, mechanistically linking forces to molecular conformations or interactions greatly benefits from real-time correlation of force and fluorescence data, yet this capability has traditionally been accessible only to a few specialists worldwide. The Lumicks-C trap is a unique commercial instrument that routinely enables concurrent sub-pN force and fluorescence measurements on biomolecular complexes.
This application brings together a diverse group of 24 academics from 4 Faculties and 8 Schools at the University of Leeds, with a shared need for force and fluorescence analyses at the single molecule level in their current and emerging research projects. Our objective is to augment our capability in single molecule analyses by the purchase of a Lumicks C-trap. The equipment will be accessible to doctoral and postdoctoral researchers, and to early career and established investigators, to further research on a wide range of topics within BBSRC remit including: protein folding and protein-protein interactions, protein-nucleic acid interactions, protein-carbohydrate interactions, biomolecular condensation and biomineralisation, cell compartmentalisation and synthetic cells, and proteins as materials.
Integration of the Lumicks C-trap into the Multi-User AFM Facility at the University of Leeds will ensure sustained technical support and infrastructure of the highest quality, facilitate the shared use of equipment and maximise synergies between the applicants’ complementary research expertise. The range of expertise spans from experimental biophysics for single-molecule force measurements and microscopy, to biochemistry to prepare ‘designer’ molecules, to theory and computational methods to guide experiments and interpret results, to the underpinning biology. The establishment of the Yorkshire Single Molecule Interest Group (YSMIG) alongside the C-Trap installation will further promote knowledge exchange and integration of single-molecule biophysics research across the region.