Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Mar 5;116(10):4031-4036.
doi: 10.1073/pnas.1814775116. Epub 2019 Feb 13.

Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression

Miranda L Jacobs  1   2 , Margrethe A Boyd  2 , Neha P Kamat  3   4   5
Affiliations

Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression

Miranda L Jacobs et al. Proc Natl Acad Sci U S A. .

Abstract

The expression and integration of membrane proteins into vesicle membranes is a critical step in the design of cell-mimetic biosensors, bioreactors, and artificial cells. While membrane proteins have been integrated into a variety of nonnatural membranes, the effects of the chemical and physical properties of these vesicle membranes on protein behavior remain largely unknown. Nonnatural amphiphiles, such as diblock copolymers, provide an interface that can be synthetically controlled to better investigate this relationship. Here, we focus on the initial step in a membrane protein's life cycle: expression and folding. We observe improvements in both the folding and overall production of a model mechanosensitive channel protein, the mechanosensitive channel of large conductance, during cell-free reactions when vesicles containing diblock copolymers are present. By systematically tuning the membrane composition of vesicles through incorporation of a poly(ethylene oxide)-b-poly(butadiene) diblock copolymer, we show that membrane protein folding and production can be improved over that observed in traditional lipid vesicles. We then reproduce this effect with an alternate membrane-elasticizing molecule, C12E8 Our results suggest that global membrane physical properties, specifically available membrane surface area and the membrane area expansion modulus, significantly influence the folding and yield of a membrane protein. Furthermore, our results set the stage for explorations into how nonnatural membrane amphiphiles can be used to both study and enhance the production of biological membrane proteins.

Keywords: cell-free protein synthesis; diblock copolymer; elastic modulus; membrane protein folding; vesicles.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Lipid vesicles improve the production of MscL during cell-free protein synthesis. (A) Schematic of a cell-free reaction in which DNA and vesicles were mixed with PURExpress kit components. (B) Schematic of the plasmid used to generate an MscLGFP fusion protein. MscL is tagged C-terminally with mEGFP: the proper folding of MscL allows GFP folding and fluorescence (Right) while the misfolding or aggregation of MscL does not permit GFP folding (Left). (C) Fluorescence of MscLGFP and soluble GFP 3.5 h after cell-free reactions with varying concentrations of DOPC vesicles, normalized to the maximum GFP fluorescence value observed for each protein. (D) Quantitative Western blot of MscLGFP from cell-free reactions shown in C. Densitometry values were normalized to reactions performed in water. ****P ≤ 0.0001 (P values were generated by ANOVA using the Dunnett test for multiple comparisons to the sample performed in water). n = 3; error bars represent standard error of the mean (SEM); ns, nonsignificant, P > 0.05.
Fig. 2.
Fig. 2.
PEO-b-PBD 1.8-kDa polymers aid MscLGFP folding. (A) Chemical structures of the amphiphiles used to generate vesicle membranes. (B) MscLGFP fluorescence after cell-free reactions with varying fractions of PEO-b-PBD 1.8 kDa in DOPC vesicles normalized to reactions performed in water. The vesicle concentration was kept constant at 10 mM total amphiphiles. n = 3; error bars represent SEM. (C) Ka values for DOPC, PEO-b-PBD, and 1:1 DOPC:PEO-b-PBD vesicles, measured via micropipette aspiration. n > 15 vesicles; error bars represent SEM. *P ≤ 0.05, nonsignificant (ns) P > 0.05 (P values were generated by ANOVA using the Dunnett test for multiple comparisons to the sample containing 0 mol% polymer).
Fig. 3.
Fig. 3.
Increasing membrane elasticity with a membrane additive improves MscLGFP folding. MscLGFP fluorescence after cell-free reactions conducted with DOPC vesicles containing varying fractions of C12E8. MscLGFP fluorescence was normalized to reactions performed in water. ***P ≤ 0.001 (P values were generated by ANOVA using the Dunnett test for multiple comparisons to the DOPC vesicle sample containing 0 mol% C12E8). n = 3; error bars represent SEM.
Fig. 4.
Fig. 4.
PEG-modified lipids decrease MscLGFP folding efficiency. (A) MscLGFP fluorescence after cell-free reactions conducted with vesicles containing 10 mol% DSPE-PEG of varying molecular weights (550, 1,000, and 2,000 Da) and 90% DOPC. (B) MscLGFP fluorescence after cell-free reactions conducted with vesicles containing 75 mol% DOPC and 25 mol% PEO-b-PBD of varying molecular weights normalized to reactions performed in water. All studies were conducted with 10 mM total concentration of amphiphiles. *P ≤ 0.05, nonsignificant (ns) P > 0.05 (P values were generated by ANOVA using the Dunnett test for multiple comparisons to the sample containing 100% DOPC in A and to the sample containing 3.5 kDa MW PEO-b-PBD in B). n = 3; error bars represent SEM.
Fig. 5.
Fig. 5.
MscLGFP folding into vesicle membranes depends on the available folding area and steric repulsion. From our experimental observations and other reported studies, pure DOPC membranes have a baseline hydrophobic area available for protein folding (A). An optimal blend of PEO-b-PBD and DOPC likely increases the available membrane area for protein folding and decreases the energy for membrane deformation (B). Membranes composed of high levels of PEO-b-PBD may inhibit protein folding into the membrane due to steric interactions or increased surface tension between PEG headgroups and the nascent protein chain (C).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Panganiban B, et al. Random heteropolymers preserve protein function in foreign environments. Science. 2018;359:1239–1243. - PubMed
    1. Allen JP. Design of energy-transducing artificial cells. Proc Natl Acad Sci USA. 2017;114:3790–3791. - PMC - PubMed
    1. Koike S, Jahn R. Probing and manipulating intracellular membrane traffic by microinjection of artificial vesicles. Proc Natl Acad Sci USA. 2017;114:E9883–E9892. - PMC - PubMed
    1. Noireaux V, Libchaber A. A vesicle bioreactor as a step toward an artificial cell assembly. Proc Natl Acad Sci USA. 2004;101:17669–17674. - PMC - PubMed
    1. Osaki T, Takeuchi S. Artificial cell membrane systems for biosensing applications. Anal Chem. 2017;89:216–231. - PubMed

Publication types

MeSH terms

LinkOut - more resources