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Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers
Yao Liu1, Elisabete C C M Moura1, Jonas M Dörr1
1Department of Membrane Biochemistry and Biophysics, Institute of Biomembranes, Utrecht University, Utrecht, the Netherlands.
Abstract:
As an integral membrane protein, purification and characterization of phospho-N- acetylmuramyl- pentapeptide translocase MraY have proven difficult. Low yield and concerns of retaining stability and activity after detergent solubilization have hampered the structure-function analysis. The recently developed detergent-free styrene-maleic acid (SMA) co-polymer system offers an alternative approach that may overcome these disadvantages. In this study, we used the detergent free system to purify MraY from Bacillus subtilis. This allowed efficient extraction of MraY that was heterologously produced in Escherichia coli membranes into SMA-wrapped nanodiscs. The purified MraY embedded in these nanodiscs (SMA-MraY) was comparable to the micellar MraY extracted with a conventional detergent (DDM) with regard to the yield and the purity of the recombinant protein but required significantly less time. The predominantly alpha-helical secondary structure of the protein in SMA-wrapped nanodiscs was also more stable against heat denaturation compared to the micellar protein. Thus, this detergent-free system is amenable to extract MraY efficiently and effectively while maintaining the biophysical properties of the protein. However, the apparent activity of the SMA-MraY was reduced compared to that of the detergent-solubilized protein. The present data indicates that this is caused by a lower accessibility of the enzyme in SMA-wrapped nanodiscs towards its polyisoprenoid substrate.
Insights
Purifying the integral membrane protein MraY is challenging. A detergent-free styrene-maleic acid (SMA) system efficiently extracts MraY into nanodiscs, preserving protein stability but reducing enzyme activity due to substrate accessibility.
Area of Science:
- Biochemistry
- Structural Biology
- Membrane Protein Research
Background:
- Purification of integral membrane proteins like MraY is difficult due to low yields and loss of stability/activity.
- Conventional detergent solubilization methods hinder structure-function analysis of MraY.
- Styrene-maleic acid (SMA) co-polymer system offers a detergent-free alternative for membrane protein purification.
Purpose of the Study:
- To evaluate the efficiency and effectiveness of the SMA system for purifying MraY from Bacillus subtilis.
- To compare the biophysical properties and activity of MraY purified using the SMA system versus conventional detergents.
- To investigate the impact of the SMA-wrapped nanodisc environment on MraY stability and function.
Main Methods:
- Heterologous production of MraY in Escherichia coli membranes.
- Extraction and purification of MraY using the detergent-free SMA co-polymer system.
- Extraction of MraY using conventional detergent (DDM) for comparison.
- Analysis of protein yield, purity, secondary structure, thermal stability, and enzyme activity.
Main Results:
- The SMA system efficiently extracted MraY into nanodiscs (SMA-MraY) with comparable yield and purity to DDM-extracted MraY, but in less time.
- SMA-MraY exhibited enhanced stability against heat denaturation compared to DDM-solubilized MraY.
- Apparent enzyme activity of SMA-MraY was reduced, attributed to lower accessibility of the polyisoprenoid substrate.
Conclusions:
- The detergent-free SMA system is an efficient method for purifying MraY while maintaining its biophysical properties.
- SMA-nanodiscs preserve MraY secondary structure and enhance thermal stability.
- Reduced activity of SMA-MraY highlights substrate accessibility as a key factor in nanodisc-based enzyme assays.
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