Current Research Areas
Single molecule tracking with nanopipette dosing
We have developed a versatile method that allows local and repeatable delivery (or depletion) of any water-soluble reagent from a nanopipette in ionic solution to make localised controlled changes in reagent concentration at a surface (Piper et al., J. Am. Chem. Soc. 2008). We will further integrate two-colour single molecule fluorescence tracking with scanning ion-conductance microscopy (SICM). A nanopipette can be used to deliver individual biomolecules through the tip of the pipette, which are then landed onto a specific site of the cell surface and bind to cell receptors, thus triggering a cascade of cell signalling events that could be followed by multi-colour single molecule tracking. This will allow the study of complex signalling networks with high spatial and temporal resolution at the single molecule level.
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Probing gene expression and gene regulation dynamics
Gene expression influences most aspects of cellular behaviour, and its variation is responsible for the phenotypic differences in a population of cells. Because DNA, RNA, key control and signalling proteins can be present and active at a few copies per cell, gene expression is an intrinsically stochastic process. We aim to use a well studied bacterial system, the E. coli Psp system, to study how gene activation process controls gene expression dynamics and movements in single living cells. We will study in vivo (a) the states of self association of PspA and its localisations, to understand how it switches from a negative regulator to an effector; (b) the localisations and dynamics of the effector protein PspG, the localisations and self associations of the transcriptional control protein PspF, and finally (c) the expression dynamics of the pspG promoter.
We are also developing a general method to determine the stoichiometry of protein complexes in living cells. The method is based on the counting of subunits of proteins by observing photobleaching steps of GFPs fused to a protein of interest. The photobleaching traces can be obtained by single molecule fluorescence imaging. We have recently developed a digital filter based on Chung-Kennedy algorithm to treat the noisy photobleaching trace and are now applying the algorithm to determine the stoichiometry of the functional PspA complexes in living bacterial cells, crucial to unravel the mechanism of bacterial stress response at the molecular level.
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DNA G-quadruplexes as novel therapeutic targets for cardiovascular disease
G-quadruplex motifs are prevalent in human genome and are enriched in regions such as telomeres and gene promoters with a strong positional bias towards the transcription start site. Recent experimental evidences suggest that formation of G-quadruplex structures within gene promoters may provide a conformational switching mechanism to influence the control of gene transcription.
Activation of members of the MAPK family has been shown to play key roles in the pathogenesis of various processes in the heart. Since traditional type-1 kinase inhibitors suffer from poor selectivity, small molecule gene regulators that act via a G-quadruplex mechanism may present attractive opportunities for the design of selective therapeutic agents that target cardiovascular disease-related genes such as MAPKs. We are working with groups of Dr. Ramon Vilar (Chemistry), Dr. Nigel Brand (NHLI) and Prof. Michael Schneider (NHLI) to employ a wide range of biochemical and biophysical techniques including single molecule FRET and fluorescence imaging to identify and distinguish possible G-quadruplex structures formed in the selected promoter sequences of the MAP4K4 gene, to investigate how specific quadruplex ligands modulate MAP4K4 gene expression in rat primary cardiac myocytes and cell lines, to characterise transcription factors interacting with the MAP4K4 promoter and to probe and locate quadruplex ligand in action. In addition, we are exploring the roles of quadruplex formation within the promoter region in the regulation of cardiac troponin I expression.
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Probing protein conformation, misfolding and aggregation by single molecule fluorescence
We have applied single molecule FRET approach to the folding mechanism of small fast folding proteins such as B-domain protein A and BBL (Fang et al., PNAS, 2007 and 2009). We have also carried out single molecule studies of the tumour suppressor p53, a transcription factor and important signalling protein related to 50% of cancers. Single molecule FRET measurements in solution have discovered the multiple conformations of full-length p53 masked by conventional techniques. (Fang et al., PNAS, Dec 2009).
Built upon these progresses, we would like to probe the misfolding and aggregation of amyloidogenic proteins in solution and in lipid membranes by single molecule fluorescence. We will use amyloid b (Ab) as a model system and develop single molecule fluorescence based approaches and theoretical tools to study the distribution of sizes of protein oligomers, follow the kinetic pathways of Ab oligomer formation, identify the key intermediates responsible for Ab aggregation, and assist in screening for small molecule inhibitors that can hinder aggregation. We would like also to probe the conformational dynamics of the unstructured reactive centre loop (RCL) of the serpin (serin protease inhibitor) and its roles in protease inhibitory function and the mechanism of misfolding of the serpin that leads to protein aggregation which causes many types of protein misfolding diseases.
