Journal article
A Novel Ultra-Stable, Monomeric Green Fluorescent Protein for Direct Volumetric Imaging of Whole Organs Using CLARITY
DJ Scott, NJ Gunn, KJ Yong, VC Wimmer, NA Veldhuis, LM Challis, M Haidar, S Petrou, RAD Bathgate, MDW Griffin
Scientific Reports | NATURE PORTFOLIO | Published : 2018
Abstract
Recent advances in thick tissue clearing are enabling high resolution, volumetric fluorescence imaging of complex cellular networks. Fluorescent proteins (FPs) such as GFP, however, can be inactivated by the denaturing chemicals used to remove lipids in some tissue clearing methods. Here, we solved the crystal structure of a recently engineered ultra-stable GFP (usGFP) and propose that the two stabilising mutations, Q69L and N164Y, act to improve hydrophobic packing in the core of the protein and facilitate hydrogen bonding networks at the surface, respectively. usGFP was found to dimerise strongly, which is not desirable for some applications. A point mutation at the dimer interface, F223D,..
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Awarded by Australian Research Council
Funding Acknowledgements
The authors would like to thank Riley Cridge, Sharon Layfield, and Gabriel Davis Jones for technical assistance, and Riley Metcalfe for protein production. D.J.S. is an NHMRC Boosting Dementia Research Leadership Fellow. R.A.D.B. is an NHMRC Senior Research Fellow. M.D.W.G is the recipient of an Australian Research Council Future Fellowship (project number FT140100544). Studies at the Florey were supported by the Victorian Government's Operational Infrastructure Support Program. Parts of this research were undertaken at the MX2 and SAXS/WAXS beam-lines of the Australian Synchrotron, Victoria, Australia. Crystallisation experiments were performed at the CSIRO Collaborative Crystallisation Centre, Melbourne, Australia.