Interactions of membrane proteins reconstituted in model membrane systems using mass spectrometry & interferometric light scattering microsopy
Lead Research Organisation:
University of Oxford
Abstract
Membrane proteins (MPs) form an essential part of life in virtually every living organism, including viruses, prokaryotes and eukaryotes. These proteins are generally of amphipathic nature, with at least a small part of the protein inserted into the hydrophobic core of the cellular membrane. They are indispensable for cell signalling, homeostasis and correct functioning of the whole organism. They also form over 50% of all current drug targets. However, studying their structure, interactions and dynamics has proven difficult due to challenges inherent to working with proteins embedded in membranes. In the past two decades, native mass spectrometry (native MS) has revolutionised our understanding of protein interactions and stability in the gas phase, enabling precise description of many protein structures and their interactions in extensive detail. Nevertheless, it remains a challenging task to characterise membrane proteins relying solely on mass spectrometry.
Interferometric light scattering microscopy (iSCAT) is a novel method that uses a single wavelength light beam to enable an all optic, label-free single molecule protein detection in the liquid phase. Additionally, it makes it possible to observe landing events dynamically over a period of time, potentially providing information regarding kinetics of protein-protein interaction, homomeric oligomer formation or subunit dissociation. Since thousands of protein landing events are observed during a single experiment, this technique also allows observation of interactions that happen too infrequently otherwise to be observed using other methods. Together, native MS and iSCAT can offer complimentary information regarding protein-protein interaction. Additionally, this combination could also shed light on the properties and behaviour of MPs in different micelle-forming detergents, liposomes, lipid nanodiscs and other systems used for solubilisation of MPs. These experimental systems are frequently used for studying MPs, but remain often poorly characterised. As such, information regarding their effects on MP behaviour and interactions could be of great help for MP research in general.
Overall, this project falls within a number of EPSRC research areas. For most part, the project falls within the Analytical Science EPSRC research area as it is mainly focused on the development of a novel combination of native MS and iSCAT. The project would first validate this combination within a known molecular biology system, such as the beta-barrel assembly machine (BAM) complex interaction with chaperone SurA and unfolded ligand proteins, such as the outer membrane protein T (OmpT). Hence, this project also falls within the Chemical Biology and Biological Chemistry EPSRC research area as it is developing a novel technology in order to progress our understanding of a biological system using the tools of physical and theoretical chemistry. Finally, this project falls within the Biophysics and Soft Matter Physics EPSRC research area due to its implementation of methodology from optics and, more generally, physics, to answer biological questions.
Interferometric light scattering microscopy (iSCAT) is a novel method that uses a single wavelength light beam to enable an all optic, label-free single molecule protein detection in the liquid phase. Additionally, it makes it possible to observe landing events dynamically over a period of time, potentially providing information regarding kinetics of protein-protein interaction, homomeric oligomer formation or subunit dissociation. Since thousands of protein landing events are observed during a single experiment, this technique also allows observation of interactions that happen too infrequently otherwise to be observed using other methods. Together, native MS and iSCAT can offer complimentary information regarding protein-protein interaction. Additionally, this combination could also shed light on the properties and behaviour of MPs in different micelle-forming detergents, liposomes, lipid nanodiscs and other systems used for solubilisation of MPs. These experimental systems are frequently used for studying MPs, but remain often poorly characterised. As such, information regarding their effects on MP behaviour and interactions could be of great help for MP research in general.
Overall, this project falls within a number of EPSRC research areas. For most part, the project falls within the Analytical Science EPSRC research area as it is mainly focused on the development of a novel combination of native MS and iSCAT. The project would first validate this combination within a known molecular biology system, such as the beta-barrel assembly machine (BAM) complex interaction with chaperone SurA and unfolded ligand proteins, such as the outer membrane protein T (OmpT). Hence, this project also falls within the Chemical Biology and Biological Chemistry EPSRC research area as it is developing a novel technology in order to progress our understanding of a biological system using the tools of physical and theoretical chemistry. Finally, this project falls within the Biophysics and Soft Matter Physics EPSRC research area due to its implementation of methodology from optics and, more generally, physics, to answer biological questions.
Organisations
| Description | Through this award, we have shown that membrane proteins and membrane mimetic systems can be studied with mass photometry. Membrane proteins are one of the most important yet challenging biological targets. They constitute about 60% of all drug targets, yet are difficult to work with due to inherent instability outside of lipid bilayer. Common solutions for this problem include membrane mimetic systems, which can add to the protein heterogeneity and impact its function. Mass photometry is a novel technique based on changes in refractive index when a biomolecule lands on a glass coverslip. This change in refractive index causes different light reflection, which interferes with the rest of the reflected light, amplifying the signal. This interference, proportional to the size of the biomolecule, is detected and converted into molecular weight. This is a unique method in its ease of use, rapidness and accuracy. It is also single molecule and label-free, which is an important characteristic for measuring biomolecules. I have shown that this method can be adapted and used for membrane proteins, showing that even well-known proteins, such as the potassium channel KcsA, behave differently when isolated from a native membrane using SMA polymers, and form dimers of tetramers as opposed to tetramers. This is important as this is the first time that we can directly observe membrane proteins in this manner, and has large implications for many of the experiments that were performed with more unnatural methods, such as detergent extraction of the membrane proteins. We have also shown that we can easily distinguish between functional and non-function preparations of lipid nanodiscs of KcsA, as confirmed by 2D NMR. Lastly, we show that this method can be applied to glycoproteins too, and by manipulation of the refractive index by glucose addition, we can make the glycan parts of glycoproteins effectively dissappear, paving the way for novel methods of glycoprotein glycan content quantification and research. |
| Exploitation Route | This funding explored the principle and basis of a large number of potential projects that will happen in future. By showing mass photometry can be used to study membrane proteins, we have enabled many new projects looking into membrane protein oligomerisation, membrane protein-soluble protein interaction, efficient drug screening procedures, but also viral spike-membrane protein interactions thanks to the projects contribution on glycoproteins. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |