Abstract
Intramembrane aspartyl proteases (IAPs) comprise one of four families of integral membrane proteases that hydrolyze substrates within the hydrophobic lipid bilayer. IAPs include signal peptide peptidase, which processes remnant signal peptides from nascent polypeptides in the endoplasmic reticulum, and presenilin, the catalytic component of the γ-secretase complex that processes Notch and amyloid precursor protein. Despite their broad biomedical reach, basic structure-function relationships of IAPs remain active areas of research. Characterization of membrane-bound proteins is notoriously challenging due to their inherently hydrophobic character. For IAPs, oligomerization state in solution is one outstanding question, with previous proposals for monomer, dimer, tetramer, and octamer. Here we used small angle neutron scattering (SANS) to characterize n-dodecyl-β-D-maltopyranoside (DDM) detergent solutions containing and absent a microbial IAP ortholog. A unique feature of SANS is the ability to modulate the solvent composition to mask all but the enzyme of interest. The signal from the IAP was enhanced by deuteration and, uniquely, scattering from DDM and buffers were matched by the use of both tail-deuterated DDM and D2O. The radius of gyration calculated for IAP and the corresponding ab initio consensus model are consistent with a monomer. The model is slightly smaller than the crystallographic IAP monomer, suggesting a more compact protein in solution compared with the crystal lattice. Our study provides direct insight into the oligomeric state of purified IAP in surfactant solution, and demonstrates the utility of fully contrast-matching the detergent in SANS to characterize other intramembrane proteases and their membrane-bound substrates.
| Original language | English |
|---|---|
| Pages (from-to) | 602-608 |
| Number of pages | 7 |
| Journal | Biophysical Journal |
| Volume | 114 |
| Issue number | 3 |
| DOIs | |
| State | Published - Feb 6 2018 |
Funding
This work was supported by a grant to R.L.L. from the National Science Foundation (NSF) (0845445). S.-H.N. acknowledges a travel grant from the College of Sciences, Georgia Tech to collect data at Oak Ridge National Laboratory. Neutron scattering studies at the CG-3 Bio-SANS instrument at the High-Flux Isotope Reactor and work in the Bio-Deuteration Laboratory of Oak Ridge National Laboratory were sponsored by the Office of Biological and Environmental Research and by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy (DOE). This work benefited from the use of the SasView application, originally developed under NSF award DMR-0520547. SasView also contains code developed with funding from the EU Horizon 2020 programme under the SINE2020 project grant 654000. This manuscript has been coauthored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the DOE. This work was supported by a grant to R.L.L. from the National Science Foundation (NSF) ( 0845445 ). S.-H.N. acknowledges a travel grant from the College of Sciences, Georgia Tech to collect data at Oak Ridge National Laboratory. Neutron scattering studies at the CG-3 Bio-SANS instrument at the High-Flux Isotope Reactor and work in the Bio-Deuteration Laboratory of Oak Ridge National Laboratory were sponsored by the Office of Biological and Environmental Research and by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy (DOE) . This work benefited from the use of the SasView application, originally developed under NSF award DMR-0520547 . SasView also contains code developed with funding from the EU Horizon 2020 programme under the SINE2020 project grant 654000 . This manuscript has been coauthored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the DOE .