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Research Interest

Investigation of activation-induced structural changes in ionotropic receptors using atomic force microscopy (AFM)

 

 

For AFM imaging, the sample is mounted on mica, and the tip is scanned across it in a raster pattern. A laser beam is bounced off the back of the cantilever and is reflected by a mirror onto a split photodiode. When the tip encounters an object on the surface of the mica, it becomes deflected, causing the position of the laser beam on the photodiode to change. The scanner moves in the Z-dimension to restore the beam to its original position. The XYZ movements of the scanner are analysed by a computer in order to generate the image. Importantly, imaging can be carried out under fluid, so that proteins can be studied under near-physiological conditions.

Several crystal and cryo-EM structures are available for ionotropic glutamate receptors (iGluRs). These studies provide fascinating 'snap-shots' of the receptors in different functional states. What is lacking, so far, is information about the kinetics underlying these structural transitions. We are using fast-scan atomic force microscopy (AFM) to study activation-induced structural changes in native, full-length iGluRs. We have found that activation triggers a rapid (<1-sec) and significant (~1-nm) reduction in the height of the extracellular domains of iGluRs integrated into supported lipid bilayers, and an increase in the relative mobility of the N-terminal domains. We are continuing to address the behaviour of iGluRs, and the effects of accessory proteins and drugs on these receptors. The figure shows the effect of activation on the height of an NMDA receptor.

Fast-scan imaging of NMDA receptor height shifts. Frames were captured at 1 Hz. A single receptor was monitored in buffer containing 100 μM glycine and 100 μM caged-glutamate. UV irradiation occurred at 107 s. A fitted Gaussian curve is overlaid on each histogram.

Protection of teeth against dental caries

Dental caries is a major public health issue worldwide, with approximately 35% of the world’s population currently suffering from dental caries of their permanent teeth. We are testing the ability of candidate proteins to protect enamel against acid-induced damage. AFM imaging is being used to screen the surface roughness of enamel samples after various treatments. We are also using AFM force spectroscopy to measure the forces of interaction of single protein molecules with enamel.  We have shown that canecystatin-5, a novel cystatin derived from sugar cane, adheres tightly to enamel, and confers significant protection against acid-induced damage. This result raises the exciting possibility that a protein derived (ironically) from sugar cane, may find a use in the fight against dental caries.

Determination of the structure and function of syncollin

Syncollin is a 16-kDa protein that was originally isolated from the exocrine pancreas. It is also expressed in epithelial cells lining the duodenum and the colon, in the spleen, and in azurophilic granules of neutrophils. It is present both free within the ZG lumen and also tightly associated with the luminal leaflet of the ZG membrane. Syncollin is secreted from both pancreatic acinar cells and from neutrophils. The expression of syncollin in the gut is almost undetectable until rats begin suckling; further, syncollin expression falls upon starvation and then rebounds upon refeeding. The syncollin gene was reported to be strongly down-regulated in the colon when a bacterial suspension was administered to germ-free mice. In addition, syncollin mRNA was significantly reduced in epithelia taken from patients with ulcerative colitis (but not Crohn’s disease). Syncollin KO mice are viable and fertile, although there is a reduction in ZG exocytosis. Syncollin levels are significantly elevated in both pancreatic juice and serum taken from pancreatic cancer patients, suggesting that it might be a useful pancreatic cancer biomarker. In addition, pan-cancer analysis of TCGA genomics data also shows syncollin to be upregulated in multiple cancer types. Syncollin is predicted to be structurally homologous to gamma-crystallin proteins, such as spherulin. Although we know a lot about it, the physiological role of syncollin is still unclear. The data summarized above could indicate a role in exocytosis, in host defence, or in cancer. We are currently addressing this issue.

Current lab members

 

PhD Student

 

 

 

 


PhD Student