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Title: Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles

Abstract

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. Lastly, in addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.

Authors:
ORCiD logo [1];  [2];  [2];  [2];  [2];  [2];  [3]
+ Show Author Affiliations
  1. National Inst. of Standards and Technology (NIST), Gaithersburg, MD (United States). Center for Neutron Research; National Inst. of Standards and Technology (NIST), Rockville, MD (United States). Inst. for Bioscience and Biotechnology Research
  2. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
  3. National Inst. of Standards and Technology (NIST), Gaithersburg, MD (United States). Center for Neutron Research; Univ. of Tennessee, Knoxville, TN (United States). Dept. of Chemistry
Publication Date:
Research Org.:
Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE; National Science Foundation (NSF); European Union (EU); National Institutes of Health (NIH)
OSTI Identifier:
1430967
Alternate Identifier(s):
OSTI ID: 1420337
Report Number(s):
LLNL-JRNL-739218
Journal ID: ISSN 0961-8368
Grant/Contract Number:  
AC52-07NA27344; DMR-1508249; DMR-0520547; 654000; R21AI120925; R01CA155642; R01GM117342
Resource Type:
Accepted Manuscript
Journal Name:
Protein Science
Additional Journal Information:
Journal Volume: 27; Journal Issue: 3; Journal ID: ISSN 0961-8368
Publisher:
The Protein Society
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; NLPs; nanodiscs; nanolipoprotein particles; SANS; SAXS; small‐angle scattering; cell-free expression

Citation Formats

Cleveland, Thomas E., He, Wei, Evans, Angela C., Fischer, Nicholas O., Lau, Edmond Y., Coleman, Matthew A., and Butler, Paul. Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles. United States: N. p., 2018. Web. doi:10.1002/pro.3365.
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Cleveland, Thomas E., He, Wei, Evans, Angela C., Fischer, Nicholas O., Lau, Edmond Y., Coleman, Matthew A., & Butler, Paul. Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles. United States. https://doi.org/10.1002/pro.3365
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Cleveland, Thomas E., He, Wei, Evans, Angela C., Fischer, Nicholas O., Lau, Edmond Y., Coleman, Matthew A., and Butler, Paul. Tue . "Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles". United States. https://doi.org/10.1002/pro.3365. https://www.osti.gov/servlets/purl/1430967.
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@article{osti_1430967,
title = {Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles},
author = {Cleveland, Thomas E. and He, Wei and Evans, Angela C. and Fischer, Nicholas O. and Lau, Edmond Y. and Coleman, Matthew A. and Butler, Paul},
abstractNote = {Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. Lastly, in addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.},
doi = {10.1002/pro.3365},
journal = {Protein Science},
number = 3,
volume = 27,
place = {United States},
year = {Tue Feb 13 00:00:00 EST 2018},
month = {Tue Feb 13 00:00:00 EST 2018}
}
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Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1002/pro.3365
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Cited by: 4 works
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Figures / Tables:

Table 1 Table 1: Nanoparticle dimensions reported by different techniques. Note in particular that DLS reports a hydrodynamic radius (Rh), which is quantitatively different, but related to, the disc diameter (D) reported here from SAXS/SANS data.
All figures and tables (6 total)
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      Figures / Tables found in this record:

      Table 1 (p. 27)table
      Figure 1 - NLP assembly methods (p. 30)figure
      Figure 2 - Size exclusion chromatography (SEC) of NLPs. (p. 31)figure
      Figure 3 - Comparison of SANS/SAXS data from different NLP preparations. (p. 32)figure
      Figure 4 - Simultaneous model fitting of SANS/SAXS data. (p. 33)figure
      Figure 5 - Fit Parameters and Uncertainty Analysis. (p. 34)figure
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