Poster Presentation The 42nd Lorne Conference on Protein Structure and Function 2017

Elucidation of Apolipoprotein-D Quaternary Structure and Dynamics using Small Angle X-ray Scattering and Hydrogen-Deuterium Exchange MS (#163)

Claudia Kielkopf 1 2 , Brett Garner 1 2 , Ganesh S Anand 3 , Simon HJ Brown 1 2
  1. School of Biology, University of Wollongong, Wollongong, NSW, Australia
  2. Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia
  3. Department of Biological Science, National University of Singapore, Singapore

Apolipoprotein-D (apoD) is a widely expressed 29 kDa glycoprotein in the lipocalin family. Cerebral apoD is protective in Alzheimer’s disease (AD) through antioxidant function and attenuation of amyloid-b pathology [1, 2]. ApoD is composed of an eight-stranded b-barrel with an adjacent a-helix. In a preformed cavity, apoD binds small hydrophobic molecules [3]. In vitro assays have identified apoD ligands including progesterone and arachidonic acid [4], however, the true physiological ligand range remains unknown. Unlike other lipocalins, apoD is referred to as monomer and dimerises upon oxidation of Met-93 during its antioxidant function [5, 6]. The native oligomeric status of apoD is unknown and it is unclear how apoD quaternary structure and dynamics change upon ligand binding. We are investigating apoD structural dynamics using two complimentary solution-based approaches. Small-angle X-Ray scattering (SAXS) creates low-resolution structural data, while hydrogen-deuterium exchange mass spectrometry (HDX-MS) probes protein dynamics through quantification of deuteron exchange in protein backbone amides.

To investigate the oligomeric state of apoD, we purified glycosylated native apoD from human breast cyst fluid and determined native size using size-exclusion chromatography (SEC), blue-native PAGE and dynamic light scattering. Results show apoD forms a predominant tetramer of ~120 kDa, challenging the view of apoD as monomer. We have established methods at the National University of Singapore to assess apoD structural dynamics by HDX-MS, achieving > 90% peptide coverage. This technique will now be used to reveal changes in apoD structure upon binding of ligands and during redox reactions that provide antioxidant activity. We are currently generating SEC-SAXS data at the Australian Synchrotron on apoD in the absence and presence of apoD ligands as this will reveal how apoD oligomerisation is influenced by ligand binding. Together, these experiments provide new insights into apoD structure and function that may eventually help to explain its protective role in AD.

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