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

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]
  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:
Report Number(s):
LLNL-JRNL-739218
Journal ID: ISSN 0961-8368
Grant/Contract Number:
AC52-07NA27344; DMR-1508249; DMR-0520547; 654000; R21AI120925; R01CA155642; R01GM117342
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
Research Org:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org:
USDOE; National Science Foundation (NSF); European Union (EU); National Institutes of Health (NIH)
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
OSTI Identifier:
1430967
Alternate Identifier(s):
OSTI ID: 1420337

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., Web. doi:10.1002/pro.3365.
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. doi:10.1002/pro.3365.
Cleveland, Thomas E., He, Wei, Evans, Angela C., Fischer, Nicholas O., Lau, Edmond Y., Coleman, Matthew A., and Butler, Paul. 2018. "Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles". United States. doi:10.1002/pro.3365.
@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 = {2018},
month = {2}
}

Works referenced in this record:

Reduction and analysis of SANS and USANS data using IGOR Pro
journal, November 2006

The 30 m Small-Angle Neutron Scattering Instruments at the National Institute of Standards and Technology
journal, June 1998
  • Glinka, C. J.; Barker, J. G.; Hammouda, B.
  • Journal of Applied Crystallography, Vol. 31, Issue 3, p. 430-445
  • DOI: 10.1107/S0021889897017020

Different Apolipoproteins Impact Nanolipoprotein Particle Formation
journal, November 2007
  • Chromy, Brett A.; Arroyo, Erin; Blanchette, Craig D.
  • Journal of the American Chemical Society, Vol. 129, Issue 46, p. 14348-14354
  • DOI: 10.1021/ja074753y

Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles
journal, March 2009
  • Blanchette, Craig D.; Cappuccio, Jenny A.; Kuhn, Edward A.
  • Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1788, Issue 3, p. 724-731
  • DOI: 10.1016/j.bbamem.2008.11.019

Self-Assembly of Discoidal Phospholipid Bilayer Nanoparticles with Membrane Scaffold Proteins
journal, August 2002
  • Bayburt, Timothy H.; Grinkova, Yelena V.; Sligar, Stephen G.
  • Nano Letters, Vol. 2, Issue 8, p. 853-856
  • DOI: 10.1021/nl025623k