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This content will become publicly available on February 13, 2019

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