Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Structural studies of HDL and applications of EM on membrane proteins
KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
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. , p. 73
Series
TRITA-STH : report, ISSN 1653-3836 ; 2017:4
Keyword [en]
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: urn:nbn:se:kth:diva-204045ISBN: 978-91-7729-339-2 (print)OAI: oai:DiVA.org:kth-204045DiVA, id: diva2:1084040
Public defence
2017-04-24, T2, Hälsovägen 11C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20170323

Available from: 2017-03-23 Created: 2017-03-23 Last updated: 2017-03-28Bibliographically approved
List of papers
1. Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs
Open this publication in new window or tab >>Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs
Show others...
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 3, article id e0152116Article in journal (Refereed) Published
Abstract [en]

An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron micros-copy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.

Place, publisher, year, edition, pages
Public Library of Science, 2016
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-185630 (URN)10.1371/journal.pone.0152116 (DOI)000372708000092 ()27010627 (PubMedID)2-s2.0-84962094377 (Scopus ID)
Funder
Swedish Research CouncilStockholm County Council
Note

QC 20160428

Available from: 2016-04-28 Created: 2016-04-25 Last updated: 2017-11-30Bibliographically approved
2. Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy
Open this publication in new window or tab >>Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy
2017 (English)In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 121, article id e55148Article in journal (Refereed) Published
Abstract [en]

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Place, publisher, year, edition, pages
Journal of Visualized Experiments, 2017
Keyword
Biochemistry, Issue 121, negative staining, protein complex, TEM, lipoxygenases, visualization, non-denaturing gel electrophoresis
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:kth:diva-204044 (URN)10.3791/55148 (DOI)000415744000005 ()28287545 (PubMedID)2-s2.0-85017206859 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20170327

Available from: 2017-03-23 Created: 2017-03-23 Last updated: 2018-01-13Bibliographically approved
3. Structural insights into substrate recognition in proton-dependent oligopeptide transporters
Open this publication in new window or tab >>Structural insights into substrate recognition in proton-dependent oligopeptide transporters
Show others...
2013 (English)In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 14, no 9, p. 804-810Article in journal (Refereed) Published
Abstract [en]

Short-chain peptides are transported across membranes through promiscuous proton-dependent oligopeptide transporters (POTs)-a subfamily of the major facilitator superfamily (MFS). The human POTs, PEPT1 and PEPT2, are also involved in the absorption of various drugs in the gut as well as transport to target cells. Here, we present a structure of an oligomeric POT transporter from Shewanella oneidensis (PepT(So2)), which was crystallized in the inward open conformation in complex with the peptidomimetic alafosfalin. All ligand-binding residues are highly conserved and the structural insights presented here are therefore likely to also apply to human POTs.

Place, publisher, year, edition, pages
Embo press, 2013
Keyword
alafosfalin, major facilitator superfamily, proton-dependent oligopeptide transporter, substrate recognition, x-ray structure
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-129104 (URN)10.1038/embor.2013.107 (DOI)000323753100013 ()2-s2.0-84883489166 (Scopus ID)
Funder
Swedish Research CouncilEU, FP7, Seventh Framework Programme
Note

QC 20130930

Available from: 2013-09-30 Created: 2013-09-19 Last updated: 2017-12-06Bibliographically approved
4. A saposin-lipoprotein nanoparticle system for membrane proteins
Open this publication in new window or tab >>A saposin-lipoprotein nanoparticle system for membrane proteins
Show others...
2016 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 13, no 4, p. 345-351Article in journal (Refereed) Published
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
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-186643 (URN)10.1038/NMETH.3801 (DOI)000374084800023 ()26950744 (PubMedID)2-s2.0-84960194524 (Scopus ID)
Note

QC 20160601

Available from: 2016-06-01 Created: 2016-05-13 Last updated: 2017-11-30Bibliographically approved
5. Structural Studies of High Density Lipoprotein by Electron Microscopy and Flexible Fitting
Open this publication in new window or tab >>Structural Studies of High Density Lipoprotein by Electron Microscopy and Flexible Fitting
(English)Manuscript (preprint) (Other academic)
National Category
Structural Biology
Research subject
Technology and Health
Identifiers
urn:nbn:se:kth:diva-204556 (URN)
Note

QC 20170328

Available from: 2017-03-28 Created: 2017-03-28 Last updated: 2017-03-30Bibliographically approved

Open Access in DiVA

fulltext(2181 kB)72 downloads
File information
File name FULLTEXT01.pdfFile size 2181 kBChecksum SHA-512
5116a3dd580866f16c224b53714fac694b79d987f9c3574e3f1d9b355cdc0582ef267e2144906a7b2dabbdc6810bafd5bf97603d5e22ffc78884c6045f0a792a
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Zhu, Lin
By organisation
Structural Biotechnology
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar
Total: 72 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 318 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf