Single Molecule Biophysics Research
Quantitative understanding of the biochemical processes taking place in living cells and organisms requires innovative physical tools that offer nanometre spatial resolution, millisecond time resolution as well as single molecule sensitivity. The main advantage of single molecule approaches is that they avoid ensemble averaging, allowing for observation of transient intermediates and heterogeneity. They are revolutionising the way many biological questions can be addressed. Our group’s interest is the development and applications of single molecule methodologies, in particular novel fluorescence imaging methods, to monitor the behaviour and properties of individual biological molecules, molecular complexes and molecular machines, both in vitro and in living cells. We are currently working in the following areas.
Single Molecule Imaging of Bacterial Stress Response
Gene expression influences most aspects of cellular behaviour, and its variation is responsible for the phenotypic differences in a population of cells. Gene expression is an intrinsically stochastic process because DNA, RNA, and some key control and signalling proteins are present and active at a few copies per cell. We aim to use a well studied bacterial system, the E. coli phage-shock-protein (Psp) system, to study how gene activation process controls gene expression dynamics and movement in single living cells. Using millisecond single molecule fluorescence microscopy in living cells we examine the localisation, dynamics and stoichiometry of the bacterial enhancer binding AAA+ ATPase protein PspF. We have established that (i) the stable repressive PspF-PspA complex is found in the nucleoid, transiently communicating with the inner membrane via PspA, (ii) two classes of nucleoid-bound PspF distinguished by their (PspA-dependent) dynamics exist under different growth conditions, (iii) PspF as a hexamer stably binds only one of the two psp promoters at a time and (iv) co-operative interactions of PspF with the basal transcription complex influence dynamics of the PspF hexamer-DNA complex.
Fig. 1 Determination of the stoichiometry of PspF complexes in living E. coli cells by single molecule fluorescence photobleaching analysis. Top: a representative photobleaching trace of a nucleoid-associated Venus-PspF complex (black) under non-stress growth condition and the corresponding filtered trace (red) based on Chung-Kennedy algorithm. The inset is a power spectrum of the Pairwise Difference Distribution Function (PDDF) of the filtered trace. Bottom: The distributions of stoichiometries and corresponding Gaussian fits from data obtained for Venus-PspF under non-stress (psp off) and stress (psp on) conditions.
DNA G-Quadruplex & Gene Regulation
DNA G-quadruplex motifs (G4s) are prevalent in human genome. They are enriched in regions such as telomeres and gene promoters. Recent experimental evidences suggest that formation of G-quadruplex structures within gene promoters may provide a conformational switching mechanism to influence gene transcription. β-adrenergic receptor (βAR) signalling pathway is directly involved in heart failure (HF). Highly conserved G-quadruplex forming sequences have been found in the promoter of genes involved in this pathway. These novel DNA structures may provide new targets for therapeutic intervention of HF. Funded by the British Heart Foundation, we are investigating the regulatory functions of these G-quadruplexes in genes of β1AR (ADRB1), adenylyl cyclase type 5 (AC5), and protein kinase A catalytic subunit a (PKACA), developing small molecule ligands that inhibit the expression of these genes in mouse/rat cell models and study the pharmacological properties of selected ligands as potential HF drugs. This research may provide an alternative genetic approach for the treatment of heart failure beyond well- known b-blockers. We are also developing single molecule experimental approaches to directly detect individual promoter quadruplexes in living cells and monitor the kinetics of single cell gene expression under the control of a DNA quadruplex switch.
Fig. 2 Mechanism of G-quadruplex (G4) binding small molecules as “genetic β-blockers” to inhibit expression of β1AR, AC5, and PKACA genes via targeting promoter and 5’-UTR quadruplexes. These small molecules can stabilise quadruplexes, and block the assembly of transcription machinery in promoter. Multiple quadruplexes around transcription start site may further increase this effect as shown in the bottom graph.
Probing Amyloid-Beta Aggregation in Physiological Conditions
The prevailing hypothesis of the cause of Alzheimer’s disease (AD), the amyloid cascade hypothesis, states that the accumulation and aggregation of amyloid-β (Aβ) peptides is a key event in the cause of the disease. The pathology of AD involves a build-up of amyloid-β peptides, predominantly of 40 and 42 residues (Aβ40 and Aβ42, respectively). This process is thought to be due to aggregation upon interactions with metal ions such as zinc and copper, eventually leading to the deposit as fibrils and plaques. Recent high profile failures in clinical trials of AD drugs, focused on Aβ production and clearance, prompted us to explore an alternative metal chaperone approach by preventing the formation of metal-laden oligomers and promoting the return of synaptic metals from oligomers back to neurons. Several fundamental biophysical questions remain to be addressed. Firstly, at what metal ion concentration and at what time do Aβ oligomers start to form in the synaptic cleft after the release of synaptic metal ions? Secondly, how do metal protein attenuating compounds (MPACs) influence the onset of the oligomerisation and what is the optimal affinity of MPAC for metal ions? And thirdly, what are the roles of the abundant cerebrospinal fluid (CSF) metal binding proteins, such as human serum albumin (HSA), in regulating the interactions between metal ions and Aβ? These questions are extremely difficult to address in vivo, due to the low nanomolar concentrations of Aβ in the CSF and the 15-20 nanometre size of the synaptic cleft. We are developing and integrating single molecule experimental and computational approaches to tackle these questions quantitatively and to provide the potential tests for intervention strategies.
Fig. 3 Roles of zinc and copper ions in synaptic transmission and the metal chaperone strategy for the treatment of AD. When Aβ interacts with these metals the peptide forms toxic oligomers and ultimately amyloid plaques. MPACs inhibit Aβ/metal interactions and promote the return of synaptic metals from toxic oligomers back to neurons, therefore facilitating normal neurotransmission.