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A saposin-lipoprotein nanoparticle system for membrane proteins
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2016 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 13, no 4, 345-351 p.Article in journal (Refereed) Published
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Text
Abstract [en]

A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptT(S02) by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016. Vol. 13, no 4, 345-351 p.
National Category
Biophysics
Identifiers
URN: urn:nbn:se:kth:diva-186643DOI: 10.1038/NMETH.3801ISI: 000374084800023PubMedID: 26950744Scopus ID: 2-s2.0-84960194524OAI: oai:DiVA.org:kth-186643DiVA: diva2:932429
Note

QC 20160601

Available from: 2016-06-01 Created: 2016-05-13 Last updated: 2017-03-27Bibliographically approved
In thesis
1. Structural studies of HDL and applications of EM on membrane proteins
Open this publication in new window or tab >>Structural studies of HDL and applications of EM on membrane proteins
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A large number of proteins interact with biological membranes, either integrated in the membrane (PepTSo2), embedded on a membrane surface (5-lipoxygenase) or encircling a cutout of lipid bilayer (apolipoprotein1 (apoA-I). They function as transporters, receptors or biocatalysts in cellular processes like inflammation or cholesterol transport which are touched upon here. Malfunction of specific membrane proteins are the cause for several diseases or disorders.

Knowledge of protein structure supports understanding of its mechanism of function. Here, transmission electron microscopy (TEM) was used for structure determination. To obtain structure information to high resolution for membrane proteins, normally surrounded by lipids, demands specific methods and materials for stabilization. Stabilized in detergent the structure of the bacterial transporter PepTSo2 was shown to form a tetramer even bound to substrate. However, with a protein based stabilizer, Salipro, the structure of PepTSo2 could be determined to high resolution.

High density lipoprotein (HDL) in blood plasma, involved in the removal of cholesterol from peripheral tissues, have a central role in cardiovascular function, metabolic syndrome and diabetes.

The HDL-particle is composed of two copies of ApoA1 and around hundred lipid molecules. From TEM data, for the first time the clearly discoidal shape could be shown by 3-dimendional reconstructions. These were used for modelling the ApoA1 protein dimer by a "biased fitting" procedure. The results indicate how ApoA1 folds around a lipid bilayer in a disc-shaped structure.

Modified HDL called nanodiscs were here used to show the Ca2+ dependent binding of 5-lipoxygenase on the nanodisc bilayer and thereby increased production of the inflammatory mediator leukotrieneA4. Dimerization of 5-lipoxygenase inactivates these functions.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2017. 73 p.
Series
TRITA-STH : report, ISSN 1653-3836 ; 2017:4
Keyword
high density lipoprotein, rHDL, apoA-I, transmission electron microscopy, membrane protein, nanodisc, Salipro, transporter
National Category
Biological Sciences
Research subject
Technology and Health
Identifiers
urn:nbn:se:kth:diva-204045 (URN)978-91-7729-339-2 (ISBN)
Public defence
2017-04-24, T2, Hälsovägen 11C, Stockholm, 10:00 (English)
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Supervisors
Note

QC 20170323

Available from: 2017-03-23 Created: 2017-03-23 Last updated: 2017-03-28Bibliographically approved

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