Invisible detergents for structure determination of membrane proteins by small‐angle neutron scattering

Abstract

A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron scattering length difference between hydrogen and deuterium. Individual hydrogen/deuterium levels of the detergent head and tail groups were achieved such that the formed micelles became effectively invisible in heavy water (D O) when investigated by neutrons. This way, only the signal from the membrane protein remained in the SANS data. We demonstrate that the method is not only generally applicable on five very different membrane proteins but also reveals subtle structural details about the sarco/endoplasmatic reticulum Ca ATPase (SERCA). In all, the synthesis of isotope-substituted detergents makes solution structure determination of membrane proteins by SANS and subsequent data analysis available to nonspecialists.

Tracking Ca 2+ ATPase intermediates in real time by x-ray solution scattering

“…Because the SERCA-containing membranes in our study were prepared at saturating levels of Ca 2+ ions (see Materials and Methods) and since all the ATP in the sample was locked in photocages, the dominating prepulse state could be assumed to be represented by [Ca 2 ]E1 structures. We also generated difference profiles using linear combinations of E1 and E2 states since small-angle neutron scattering (SANS) data using detergents with deuteration levels optimized to provide an invisible background in a calcium-free D 2 O buffer were shown to match best with a linear combination of Ca 2+ -free E1 and E2 SERCA states in solution (35). However, in no case were the linear combinations superior to the fits resulting from assuming [Ca 2 ]E1 [Protein Data Bank (PDB) ID: 2C9M] as sole component in the prepulse solution (table S1), which also is consistent with the experimental conditions with excess Ca 2+ to saturate the binding sites.…”

Combining molecular dynamics simulations with small-angle X-ray and neutron scattering data to study multi-domain proteins in solution

“…An effective way to increase the signal-to-noise ratio in SANS is to minimize the incoherent background scattering from H2O in the sample. A relevant contrast situation is therefore obtained at 100% D2O, where the signal-to-noise ratio can be improved radically, and data quality comparable with SAXS data can be obtained, even for challenging protein systems that are difficult to express in large quantities (52). Moreover, to complement the SAXS, one out of three domains should ideally be matched out, in order to have a contrast situation where only two domains contribute to the total scattering ( Fig.…”

Assessment of structure factors for analysis of small-angle scattering data from desired or undesired aggregates

“…Examples similar to Examples 4 and 5 can be found in our previous work (Midtgaard et al, 2018;Larsen, Dorosz et al, 2018;Martin et al, 2019), where real SANS data on integral membrane proteins were analysed using structure factors to 'filter out' aggregates in the analysis.…”

“…1, D ¼ 2 and D ¼ 3. S 2 ðqÞ was used byMalik et al (2013) to account for aggregates in samples of coiled coil peptides and byMidtgaard et al (2018) to fit SANS data of a sample of the membrane protein transporter sarco/endoplasmic reticulum calcium ATPase (SERCA). S 2 ðqÞ was also used by to describe SANS data measured on samples of the AMPA-type glutamate receptor 2 (GluA2, an ion channel membrane protein) as the samples contained minor fractions of oligomeric aggregates.…”