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 biology of integral membrane proteins - From methods to molecular mechanisms
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Pär Nordlund)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins are vital components in the cell and crucial for the proliferation of all living organisms. Unfortunately our collective knowledge of structures of membrane proteins is very limited, as compared to the information available on soluble proteins. This is to a large extent due to the outstanding challenge of working with membrane proteins and the relatively high cost associated with determining a membrane protein structure.  Therefore, the establishment of efficient methods and means for the production and crystallization of membrane proteins is urgently needed. The two methods explored in this thesis  are aimed to achieve rapid optimization of expression and purification conditions of membrane proteins, thereby allowing for the rapid production of more suitable samples for crystallization trials.

Despite the challenges in membrane protein structure determination two structures are presented in the thesis:

The first structure determined is of the CorA magnesium transporter from Thermotoga maritima will be the focus of this thesis. The CorA revealed a pentameric protein in a closed state. The presence of two regulatory metal binding sites is suggested, as well as a putative magnesium ion bound in the ion conductive pathway.

The second structure is of the human enzyme LTC4-synthase, which catalyzes the pivotal step in eicosanoid synthesis by the conjugation of glutathione to LTA4, a reactive epoxide-containing derivative from arachidonic acid. The products of this step, the so-called cysteinyl leukotrienes are potent inflammatory mediators making this enzyme a potential drug target. The structure reveals a charged binding pocket for a horseshoe-shaped glutathione, and a hydrophobic binding pocket for a lipophilic LTA4 molecule. Based on the structure a key residue for catalysis has been identified, Arg 104, which is proposed to play a critical role in activating the thiol group of glutathione for the nucleophilic attack on LTA4.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm Univeristy , 2009. , 59 p.
Keyword [en]
membrane proteins, CorA, magnesium transport, screening, Leukotriene C4 synthase, detergents
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-30069ISBN: 978-91-628-7899-3 (print)OAI: oai:DiVA.org:su-30069DiVA: diva2:241167
Public defence
2009-10-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2009-10-07 Created: 2009-10-01 Last updated: 2012-08-10Bibliographically approved
List of papers
1. A simple strategy towards membrane protein purification and crystallization
Open this publication in new window or tab >>A simple strategy towards membrane protein purification and crystallization
2006 (English)In: International Journal of Biological Macromolecules, ISSN 0141-8130, E-ISSN 1879-0003, Vol. 39, no 1-3, 83-87 p.Article in journal (Refereed) Published
Abstract [en]

A simple and cost-efficient detergent screening strategy has been developed, by which a number of detergents were screened for their efficiency to extract and purify the recombinant ammonium/ammonia channel, AmtB, from Escherichia coli, hence selecting the most efficient detergents prior to large-scale protein production and crystallization. The method requires 1 ml cell culture and is a combination of immobilized metal ion affinity chromatography and filtration steps in 96-well plates. Large-scale protein purification and subsequent crystallization screening resulted in AmtB crystals diffracting to low resolution with three detergents. This strategy allows exclusion of detergents with the lowest probability in yielding protein crystals and selecting those with higher probability, hence, reducing the number of detergents to be screened prior to large-scale membrane protein purification and perhaps also crystallization.

Keyword
membrane proteins, detergent screen, high-throughput
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Structural Biology
Identifiers
urn:nbn:se:su:diva-29986 (URN)10.1016/j.ijbiomac.2006.02.011 (DOI)
Available from: 2009-10-01 Created: 2009-09-27 Last updated: 2017-12-13Bibliographically approved
2. Expression and purification of the recombinant membrane protein YidC: A case studyfor increased solubility and stability
Open this publication in new window or tab >>Expression and purification of the recombinant membrane protein YidC: A case studyfor increased solubility and stability
2008 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 62, no 1, 49-52 p.Article in journal (Refereed) Published
Abstract [en]

YidC is an inner membrane protein from Escherichia coli and is an essential component in insertion, trans- location and assembly of membrane proteins in the membranes. Previous purification attempts resulted in heavy aggregates and precipitated protein at later stages of purification. Here we present a rapid and straightforward stability screening strategy based on gel filtration chromatography, which requires as little as 10 lg of protein and takes less than 15 min to perform. With this technique, we could rapidly screen several buffers in order to identify an optimum condition that stabilizes purified YidC. After optimization we could obtain several milligrams of purified YidC that could be easily prepared at high con- centrations and that was stable for weeks at +4 C. The isolated protein is thus well suited for structural studies.

Keyword
Membrane proteins insertion system, stability optimization, analytical size exclusion chromatography, gel filtration, buffer screen
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biogeochemistry
Identifiers
urn:nbn:se:su:diva-29987 (URN)10.1016/j.pep.2008.05.011 (DOI)000260061900008 ()
Available from: 2009-10-01 Created: 2009-09-27 Last updated: 2017-12-13Bibliographically approved
3. Crystal structure of a divalent metal ion transporter CorA at 2.9 angstrom resolution
Open this publication in new window or tab >>Crystal structure of a divalent metal ion transporter CorA at 2.9 angstrom resolution
Show others...
2006 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 313, no 5785, 354-357 p.Article in journal (Refereed) Published
Abstract [en]

CorA family members are ubiquitously distributed transporters of divalent metal cations and are considered to be the primary Mg2 transporter of Bacteria and Archaea. We have determined a 2.9 angstrom resolution structure of CorA from Thermotoga maritima that reveals a pentameric cone– shaped protein. Two potential regulatory metal binding sites are found in the N-terminal domain that bind both Mg2+ and Co2+. The structure of CorA supports an efflux system involving dehydration and rehydration of divalent metal ions potentially mediated by a ring of conserved aspartate residues at the cytoplasmic entrance and a carbonyl funnel at the periplasmic side of the pore.

Keyword
Magnesium, cora, transporter, channel, membrane proteins
National Category
Biochemistry and Molecular Biology
Research subject
Structural Biology
Identifiers
urn:nbn:se:su:diva-29989 (URN)10.1126/science.1127121 (DOI)
Available from: 2009-10-01 Created: 2009-09-27 Last updated: 2017-12-13Bibliographically approved
4. Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase
Open this publication in new window or tab >>Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase
2007 (English)In: Nature, ISSN 0028-0836, Vol. 448, no 7153, 613-616 p.Article in journal (Refereed) Published
Abstract [en]

Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A4 (LTA4). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C4 (LTC4) in a reaction catalysed by LTC4 synthase: this reaction is the key step in cysteinyl leukotriene formation. Here we present the crystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 A resolution, respectively. The structure reveals a homotrimer, where each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a horseshoe-shaped conformation on GSH, and effectively positions the thiol group for activation by a nearby arginine at the membrane-enzyme interface. In addition, the structure provides a model for how the omega-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular 'ruler' to align the reactive epoxide at the thiol of glutathione. This provides new structural insights into the mechanism of LTC4 formation, and also suggests that the observed binding and activation of GSH might be common for a family of homologous proteins important for inflammatory and detoxification responses.

National Category
Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-27058 (URN)10.1038/nature06009 (DOI)000248446700048 ()
Note
Totalt antal författare: 10Available from: 2009-04-22 Created: 2009-04-22 Last updated: 2009-04-23Bibliographically approved

Open Access in DiVA

fulltext(11971 kB)1055 downloads
File information
File name FULLTEXT01.pdfFile size 11971 kBChecksum SHA-512
43f6d6c8a6910a5420fe9045183f5c9086c03da51feaffaf16b76d445490a2147dfe5062106b19daee4e7cf934e6e3edc8ac18d8e6bffbfc397c7f00d2fe6735
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Niegowski, Damian
By organisation
Department of Biochemistry and Biophysics
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Search outside of DiVA

GoogleGoogle Scholar
Total: 1055 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: 196 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