Abstract
Background
Small angle scattering techniques are beginning to be more widely utilised for structural analysis of biological systems. However, applying these techniques to study membrane proteins still remains problematic, due to sample preparation requirements and analysis of the resulting data. The development of styrene-maleic acid co-polymers (SMA) to extract membrane proteins into nanodiscs for further study provides a suitable environment for structural analysis.
Methods
We use small angle neutron scattering (SANS) with three different contrasts to determine structural information for two different polymer nanodisc-incorporated proteins, Outer membrane protein F (OmpF) and gramicidin. Ab initio modelling was applied to generate protein/lipid structures from the SANS data. Other complementary structural methodologies, such as DLS, CD and TEM were compared alongside this data with known protein crystal structures.
Results
A single-phase model was constructed for gramicidin-containing nanodiscs, which showed dimer formation in the centre of the nanodisc. For OmpF-nanodiscs we were able to construct a multi-phase model, providing structural information on the protein/lipid and polymer components of the sample.
Conclusions
Polymer-nanodiscs can provide a suitable platform to investigate certain membrane proteins using SANS, alongside other structural methodologies. However, differences between the published crystal structure and OmpF-nanodiscs were observed, suggesting the nanodisc structure could be altering the folding of the protein.
General significance
Small angle scattering techniques can provide structural information on the protein and polymer nanodisc without requiring crystallisation of the protein. Additional complementary techniques, such as ab initio modelling, can generate alternative models both the protein and nanodisc system.
Small angle scattering techniques are beginning to be more widely utilised for structural analysis of biological systems. However, applying these techniques to study membrane proteins still remains problematic, due to sample preparation requirements and analysis of the resulting data. The development of styrene-maleic acid co-polymers (SMA) to extract membrane proteins into nanodiscs for further study provides a suitable environment for structural analysis.
Methods
We use small angle neutron scattering (SANS) with three different contrasts to determine structural information for two different polymer nanodisc-incorporated proteins, Outer membrane protein F (OmpF) and gramicidin. Ab initio modelling was applied to generate protein/lipid structures from the SANS data. Other complementary structural methodologies, such as DLS, CD and TEM were compared alongside this data with known protein crystal structures.
Results
A single-phase model was constructed for gramicidin-containing nanodiscs, which showed dimer formation in the centre of the nanodisc. For OmpF-nanodiscs we were able to construct a multi-phase model, providing structural information on the protein/lipid and polymer components of the sample.
Conclusions
Polymer-nanodiscs can provide a suitable platform to investigate certain membrane proteins using SANS, alongside other structural methodologies. However, differences between the published crystal structure and OmpF-nanodiscs were observed, suggesting the nanodisc structure could be altering the folding of the protein.
General significance
Small angle scattering techniques can provide structural information on the protein and polymer nanodisc without requiring crystallisation of the protein. Additional complementary techniques, such as ab initio modelling, can generate alternative models both the protein and nanodisc system.
| Original language | English |
|---|---|
| Article number | 100033 |
| Journal | BBA Advances |
| Volume | 2 |
| Early online date | 16 Dec 2021 |
| DOIs | |
| Publication status | Published - Jan 2022 |
Bibliographical note
Funding Information:K.A.M would like to thank the STFC (ISIS) and EPSRC for co-funding a PhD studentship via the EPSRC Centre for Doctoral Training in Sustainable Chemical Technologies (EP/L016354/1; STFC Studentship Agreement no. #SA-54). A.D is funded by the Annett Trust and the British Council Newton Fund. The authors would like to acknowledge Dr Leide Cavalcanti and Dr Marcelo Alves Da Silva for assisting with the research conducted at ISIS Neutron and Muon Source (Harwell, UK) (experiment RB1920099, DOI: 10.5286/ISIS.E.RB1920099). We would like to further acknowledge Diana Lednitzky, for the sample preparation and in obtaining the TEM images in this manuscript.
Keywords
- Ab initio
- MONSA
- Nanodisc
- Outer membrane protein F
- SMALP
- Small angle neutron scattering
ASJC Scopus subject areas
- Biochemistry
- Biophysics
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Dataset for "Ab initio reconstruction of small angle scattering data for membrane proteins in copolymer nanodiscs"
Morrison, K. (Creator), Whitley, P. (Creator), Price, G. (Creator), Edler, K. (Creator) & Doutch, J. (Data Collector), University of Bath, 16 Dec 2021
DOI: 10.15125/BATH-01066
Dataset