Preparation of a Deuterated Membrane Protein for Small-Angle Neutron Scattering

Yuqi Wu; Kevin L. Weiss; Raquel L. Lieberman

doi:10.1007/978-1-0716-1394-8_12

Preparation of a Deuterated Membrane Protein for Small-Angle Neutron Scattering

Yuqi Wu, Kevin L. Weiss, Raquel L. Lieberman

Biological Labeling and Scattering

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

1 Scopus citations

Abstract

This chapter outlines a protocol developed to prepare a purified deuterated membrane protein for a small-angle neutron scattering (SANS) experiment. SANS is a noninvasive technique well suited to studying membrane protein solution structures, and deuteration enhances the signal from the protein over the background (Breyton et al., Eur Phys J E Soft Matter 36 (7):71, 2013; Garg et al., Biophys J 101 (2):370–377, 2011). We present our workflow: transformation of our plasmid into E. coli, cell growth and expression of our deuterated protein, membrane isolation, detergent solubilization, protein purification, purity assessment, and final preparation for SANS.

Original language	English
Title of host publication	Methods in Molecular Biology
Publisher	Humana Press Inc.
Pages	219-235
Number of pages	17
DOIs	https://doi.org/10.1007/978-1-0716-1394-8_12
State	Published - 2021

Publication series

Name	Methods in Molecular Biology
Volume	2302
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Funding

SANS studies on IAPs in the Lieberman lab are supported by NSF grant 1817796. The Office of Biological and Environmental Research supported research at Oak Ridge National Laboratory’s Center for Structural Molecular Biology (CSMB), using facilities supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy.

Funders	Funder number
Oak Ridge National Laboratory
National Science Foundation	1817796
U.S. Department of Energy
Basic Energy Sciences
Canadian Society for Molecular Biosciences

Keywords

Contrast match point
Detergent
Deuteration
Intramembrane proteolysis
Membrane protein
Neutron scattering
Recombinant expression
α-Helix

Access to Document

10.1007/978-1-0716-1394-8_12

Cite this

@inbook{1ffa2e02eca844bab5a296c9d33533d0,

title = "Preparation of a Deuterated Membrane Protein for Small-Angle Neutron Scattering",

abstract = "This chapter outlines a protocol developed to prepare a purified deuterated membrane protein for a small-angle neutron scattering (SANS) experiment. SANS is a noninvasive technique well suited to studying membrane protein solution structures, and deuteration enhances the signal from the protein over the background (Breyton et al., Eur Phys J E Soft Matter 36 (7):71, 2013; Garg et al., Biophys J 101 (2):370–377, 2011). We present our workflow: transformation of our plasmid into E. coli, cell growth and expression of our deuterated protein, membrane isolation, detergent solubilization, protein purification, purity assessment, and final preparation for SANS.",

keywords = "Contrast match point, Detergent, Deuteration, Intramembrane proteolysis, Membrane protein, Neutron scattering, Recombinant expression, α-Helix",

author = "Yuqi Wu and Weiss, {Kevin L.} and Lieberman, {Raquel L.}",

note = "Publisher Copyright: {\textcopyright} 2021, Springer Science+Business Media, LLC, part of Springer Nature.",

year = "2021",

doi = "10.1007/978-1-0716-1394-8_12",

language = "English",

series = "Methods in Molecular Biology",

publisher = "Humana Press Inc.",

pages = "219--235",

booktitle = "Methods in Molecular Biology",

}

TY - CHAP

T1 - Preparation of a Deuterated Membrane Protein for Small-Angle Neutron Scattering

AU - Wu, Yuqi

AU - Weiss, Kevin L.

AU - Lieberman, Raquel L.

PY - 2021

Y1 - 2021

N2 - This chapter outlines a protocol developed to prepare a purified deuterated membrane protein for a small-angle neutron scattering (SANS) experiment. SANS is a noninvasive technique well suited to studying membrane protein solution structures, and deuteration enhances the signal from the protein over the background (Breyton et al., Eur Phys J E Soft Matter 36 (7):71, 2013; Garg et al., Biophys J 101 (2):370–377, 2011). We present our workflow: transformation of our plasmid into E. coli, cell growth and expression of our deuterated protein, membrane isolation, detergent solubilization, protein purification, purity assessment, and final preparation for SANS.

AB - This chapter outlines a protocol developed to prepare a purified deuterated membrane protein for a small-angle neutron scattering (SANS) experiment. SANS is a noninvasive technique well suited to studying membrane protein solution structures, and deuteration enhances the signal from the protein over the background (Breyton et al., Eur Phys J E Soft Matter 36 (7):71, 2013; Garg et al., Biophys J 101 (2):370–377, 2011). We present our workflow: transformation of our plasmid into E. coli, cell growth and expression of our deuterated protein, membrane isolation, detergent solubilization, protein purification, purity assessment, and final preparation for SANS.

KW - Contrast match point

KW - Detergent

KW - Deuteration

KW - Intramembrane proteolysis

KW - Membrane protein

KW - Neutron scattering

KW - Recombinant expression

KW - α-Helix

UR - http://www.scopus.com/inward/record.url?scp=85104721910&partnerID=8YFLogxK

U2 - 10.1007/978-1-0716-1394-8_12

DO - 10.1007/978-1-0716-1394-8_12

M3 - Chapter

C2 - 33877630

AN - SCOPUS:85104721910

T3 - Methods in Molecular Biology

SP - 219

EP - 235

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -

Preparation of a Deuterated Membrane Protein for Small-Angle Neutron Scattering

Abstract

Publication series

Funding

Keywords

Access to Document

Other files and links

Fingerprint

Cite this