Biology subjects

Aspinwall, C. A.

Publications and source records attributed to Aspinwall, C. A..

3 recordsLinked to original sources

bioRxiv2023

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry (MS) in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for mechanisms of their biological functions as well as their potential as therapeutic targets.

biophysics↗

bioRxiv2023

Characterizing Adeno-Associated Virus Capsids with both Denaturing and Intact Analysis Methods

Adeno-associated virus (AAV) capsids are among the leading gene delivery platforms used to treat a vast array of human diseases and conditions. AAVs exist in a variety of serotypes due to differences in viral protein (VP) sequences, with distinct serotypes targeting specific cells and tissues. As the utility of AAVs in gene therapy increases, ensuring their specific composition is imperative for correct targeting and gene delivery. From a quality control perspective, current analytical tools are limited in their selectivity for viral protein (VP) subunits due to their sequence similiaries, instrumental difficulties in assessing the large molecular weights of intact capsids, and the uncertainity in distinguishing empty and filled capsids. To address these challenges, we combine two distinct analytical workflows that assess the intact capsids and VP subunits separately. First, charge detection-mass spectrometry (CD-MS) was applied for characterization of the intact capsids and then liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) separations were used for capsid denaturing measurements. This multi-method combination was applied to 3 AAV serotypes (AAV2, AAV6, and AAV8) to evaluate their intact empty and filled capsid ratios and then examine the distinct VP sequences and modifications present.

biochemistry↗

bioRxiv2023

Identifying Membrane Protein-Lipid Interactions with Lipidomic Lipid Exchange-Mass Spectrometry

Lipids can play important roles in modulating membrane protein structure and function. However, it is challenging to identify natural lipids bound to membrane proteins in complex bilayers. Here, we developed lipidomic lipid exchange-mass spectrometry (LX-MS) to study the lipid affinity for membrane proteins on a lipidomic scale. We first mix membrane protein nanodiscs with empty nanodiscs that have no embedded membrane proteins. After allowing lipids to passively exchange between the two populations, we separate the two types of nanodiscs and perform lipidomic analysis on each with liquid chromatography and mass spectrometry. Enrichment of lipids in the membrane protein nanodiscs reveals the affinity of individual lipids for binding the target membrane protein. We apply this approach to study three membrane proteins. With the E. coli ammonium transporter AmtB and aquaporin AqpZ in nanodiscs with E. coli polar lipid extract, we detected binding of cardiolipin and phosphatidyl-glycerol lipids to the proteins. With the acetylcholine receptor in nanodiscs with brain polar lipid extract, we discovered a complex set of lipid interactions that depended on the head group and tail composition. Overall, lipidomic LX-MS provides a detailed understanding of the lipid binding affinity and thermodynamics for membrane proteins in complex bilayers and provides a unique perspective on the chemical environment surrounding membrane proteins.

biophysics↗