Institute of Biological and Biomedical Sciences
The McGeehan Lab is one of the five groups in the Molecular Biophysics division of the Institute of Biomedical and Biomolecular Science (IBBS) at the University of Portsmouth. The group focuses on the structure and function of proteins, nucleic acids and their complexes. We combine X-ray crystallography with other biophysical methods such as spectroscopy, analytical ultracentrifugation and small-angle X-ray (SAXS) and neutron scattering (SANS). We also have strong collaborations in other fields including electron microscopy and molecular dynamic simulations.
For further details and publication history please see my staff profile
Our current laboratory research
Protein-nucleic acid interactions
We work on the mechanisms that control gene expression, in particular, the selective binding of transcriptional regulator proteins to their cognate DNA control sequences. We have mapped and characterised binding sites for a range transcriptional regulators and solved the 3D structures of the proteins, the nucleic acids and the bound complexes by X-ray crystallography.
Over the past decade, we have focused on the controller proteins, or C-proteins, that control the expression of restriction-modification (R-M) systems in bacteria. Likened to an ancient form of innateimmunity, bacteria use the R-M system to protect themselves against invading viruses. The system first tags the host DNA and then produces an enzyme which destroys un-tagged infecting viral DNA. The system works by employing an elegant genetic switch that controls the timing of expression for each component. A combination of structural and biophysical approaches has revealed intricate details in the mechanisms that underpin DNA recognition.
Mitochondrial-encoded proteins in disease
Mitochondria are the energy producing organelles of the cell, and mutations within their genome can cause numerous and often severe human diseases. At the heart of every mitochondrion is a set of five large multi-protein machines collectively known as the mitochondrial respiratory chain (MRC).This cellular machinery is central to several processes important for maintaining homeostasis within cells, including the production of ATP. The MRC is unique due to the bigenomic origin of its interacting proteins, and this complexity makes the prediction of function and pathological outcome from primary sequence mutation data extremely challenging.
Our recent work demonstrates how 3D structural analysis can be employed to predict the functional importance of mutations in mtDNA protein-coding genes. We have generated a 3D disease map of mitochondrial proteins and revealed a new class of mutation site not recognised as pathogenic by current approaches. Given the general lack of any successful therapeutic approaches for disorders of the MRC, these findings may inform the development of new diagnostic and prognostic biomarkers, as well as new drugs and targets for gene therapy.
Nucleosome Assembly Proteins
Histone chaperones direct the productive assembly and disassembly of nucleosomes by facilitating histone deposition and exchange. They are therefore central to the regulation of multiple cellular processes, such as chromatin remodeling, cell cycle control, DNA replication, and transcription. The nucleosome assembly protein (NAP) family represents a key group of histone chaperones that are essential for cell viability. Several x-ray structures of NAP1 dimers are available; however, there are currently no structures of this ubiquitous chaperone in complex with histones.
We have characterized NAP1 from Xenopus laevis and reveal that it forms discrete multimers with histones H2A/H2B and H3/H4 at a stoichiometry of one NAP dimer to one histone fold dimer. These complexes have been characterized by size exclusion chromatography, analytical ultracentrifugation, multiangle laser light scattering, and small-angle x-ray scattering to reveal their oligomeric assembly states in solution. By employing single-particle cryo-electron microscopy, we visualized these complexes for the first time and show that they form heterogeneous ring-like structures, potentially acting as large scaffolds for histone assembly and exchange.
X-ray Radiation Damage to Biological Macromolecules
X-ray radiation-induced damage (XRID) in biological macromolecules presents a significant challenge in macromolecular crystallography (MX), but could also provide a novel platform for a more rational approach to some radiation-based cancer therapies. Studies combining monochromatic X-ray radiation therapy with the cancer chemotherapy agent Cisplatin have demonstrated a synergistic therapeutic effect, but the underlying mechanisms remain elusive. Despite significant progress in DNA radiochemistry, it remains unclear how radiation interacts and causes specific damage to biologically relevant complexes.
Since DNA is rarely naked in the cell, we have undertaken XRID studies on crystals of nucleoprotein complexes. Using dose-dependent high-resolution crystallography we have been able to map the specific XRID sites in both protein-DNA and protein-RNA complexes. The results support solution binding data and indicate that the nucleic acid components are far more radiation resistant than the protein components within the complexes. These data provide a starting point to investigate the radiation chemistry that underpins this partitioning effect and may lead to a better understanding of the interaction of biological tissues with radiation.
Novel cellulose degrading enzymes
Cellulose is the most abundant biopolymer on earth and is composed entirely of glucose. Cellulose isoften trapped in the highly recalcitrant material lignocellulose making it an expensive substrate for bioethenol production. The current stock of enzymes used within cellulolytic cocktails are primarily derived from terrestrial bacteria & fungi, however, they tend to lose activity in the high salt conditions used to break apart lignocellulose during industrial processing.
We have recently solved the X-ray crystal structures of a novel family 7 glycoside hydrolase (GH) enzyme found in the gut of the marine crustacean Limnoria quadripunctata. Other woodborers, suchtermites for example, employ a community of bacteria and protists to digest wood. Limnoria is quite different, instead possessing a sterile gut, and producing all the necessary enzymes for digestion endogenously. It has been likened to a natural bioreactor. Biophysical and biochemical data revealed that this enzyme is highly salt tolerant and molecular dynamics simulations demonstrated that the enzyme is highly flexible compared to other GH7 enzymes with novel features that make it attractive from an industrial biofuels perspective.
Prion capture and Gene 5 Pr
Single-stranded DNA-binding proteins constitute an important class of DNA-binding protein, playing a key role in DNA replication, transcription and translation. The gene 5 protein (g5p) of filamentous bacteriophage fd is the most widely studied of the ssDNA-binding proteins and is responsible for the switch from double-stranded to single-stranded viral DNA replication following infection of the bacterial cell. Our lab is working on the higher-order complexes between g5p and nucleic acid structures such as G-quartets.
Collaboration with Dr Wenquan Zou at the Department of Pathology, Case Western Reserve University School of Medicine, USA showed that g5p has an unusually high affinity for the infective form of the prion protein PrP. The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrP(C) into its pathogenic isoform PrP(Sc 1). PrP(C) is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrP(Sc) forms detergent-insoluble aggregates and is partially resistant to PK. The conversion of PrP(C)to PrP(Sc) is known to involve a conformational transition of α-helical to β-sheet structures of the protein, however, the in vivo pathway is still poorly understood. The use of g5p to selectively enrich samples of infected brain tissue for PrP(Sc) has proved to be extremely useful for the identification and analysis of prion-related diseases.