We are part of the Astbury Centre for Structural Molecular Biology at the University of Leeds and make full use of the available facilities, including NMR, mass spectrometry, PELDOR, Cryo-EM, crystallography, SEC-MALLS, FRET and Molecular Dynamics simulations, as well as the many well placed collaborators across biology, chemistry and physics.
We are proud of our extensive network of internal and external collaborators, especially those from the EU and further afield. These links allow our research to extend beyond the scope of solely structural biology, but also into synthetic chemistry to biophysical investigations to cellular biology to plant sciences. Thereby facilitating our engagement with all sizes of study: from small molecules up to entire organisms.
Our major projects include:
GPCRs
G-Protein Coupled Receptors are one of the most widely studied family of membrane proteins and some of the most common drug targets. Our contribution to the study of these proteins covers most routes of investigation and are heavily linked to industry. We have worked towards novel structures, stabilisation of constructs, and small molecule co-crystal structures to guide drug design and discovery, using techniques such as crystallography, functional assays, PELDOR, stability testing through melting curves and nanoDSF. We make use of the different advances in GPCR study, such as nanobodies, mini-G and G-protein complexes. This research is primarily led by Claudia and Jamie with our collaborators: Dr Dan Donnelly, Dr Veli-Pekka Jaakola at ConfoTherapeutics and Novartis, part of the RAMP ITN.
Transporters
Transporters are drug targets for a several applications, including drug resistance, cancer and virus response. Therefore, the Goldman Lab is dedicated to structurally and functionally characterising transporters primarily from bacteria, fungi and mammals. Our prokaryotic transporter families of interest are the Major Facilitator Superfamily, which is being led by Ana, and Proteobacterial Antimicrobial Compound Efflux transporters, which is being led by Maria. We are interested in bacterial adhesion proteins, in particular trimeric autotransporters from gram negative bacteria. These structural studies are led by Anchal and Andreas. In terms of eukaryotic work, we are interested in Equilibrative Nucleoside Transporters and the ATP-Binding Cassette Superfamily, which are led by Jess and Jack, respectively. Our structural characterisation is achieved through electron microscopy and crystallography, whereas the functional investigations are achieved through radiolabelled and fluorescent transport, binding and whole-cell accumulation assays, which are typically carried out in liposomes to allow us to study these systems in their natural lipidated environment. These investigations are in collaboration with Prof Henderson, Dr Postis, Prof Baker, Dr Muench, Dr Hassan and Dr Paulsen.
Membrane Integral Pyrophosphatases
Membrane integral pyrophosphatases are clinically and agriculturally relevant enzymes that couple the break down of pyrophosphate to the pumping of a cation across a membrane. These are validated drug targets and their major targets are protozoan parasites. The investigation in the Goldman Lab orientates around gaining an in-depth structural and mechanistic understanding of these enzymes. This is being achieved through traditional and serial crystallography, enzymatic and cation pumping activity testing, small molecule screening in vitro and in silico, and dynamic measurements in silico using Molecular Dynamics simulations and in vitro using PELDOR and FRET. These studies are performed in the UK by Jannik and Alex and in Helsinki by Keni. We work closely with Dr Kalli, Dr Tuma, Prof Fishwick, Dr Pliotas, and Molecular Dimensions.
Receptor Tyrosine Kinases
Tyrosine kinases are, much like GPCRs, a widely studied family of membrane protein receptor. We are interested in RET and the Fibroblast Growth Factor Receptor, which play crucial roles in a variety of processes, including obesity, cancer, embryonic development and wound repair, via their activation of signalling cascades in the cell. The major focus of our investigations is understanding the complexes these receptors form and how these influence and regulate their activity. For RET, the complex we are interested in is with GDF15 and GFRAL, which is being led by Yixin. Whereas for FGFR, we are interested in the receptor in complex with chaperones Hsp90 and Cdc37, which Dr Brendan Farrell detailed in his thesis. This structural work takes full advantage of the Cryo-EM facilities we have access to, as well as NMR. We work on these projects in collaboration with Prof Breeze and Dr Muench.
Approaches to Improving Membrane Protein Structural Studies
As well as these highly specific projects, we are also heavily invested in improving the ease of membrane protein study. Membrane proteins represent a huge amount of the genome and yet only a fraction of the available solved structures are membrane proteins. This is for a variety of reasons: low expression level, solubility, purification contaminants, inherent instability and conformational flexibility.
We are developing cutting edge approaches to reduce these issues. For instance, one of the most common contaminants following IMAC purification from E. coli is AcrB, which we are trying to reduce through synthetic biology techniques to create more “protein purification friendly” E. coli strains. We have also developed a novel pipeline IMPROvER to identify stabilising mutations that has proved highly successful across four in-lab case studies and is now being applied to industrial targets. To make our investigations in each of these areas more efficient, we employ fractional factorial workflows to these processes, to save time and materials.