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
Conformational changes of recombinant Ca2+-ATPase studied by reaction-induced infrared difference spectroscopy
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. chenge.li@dbb.su.se.
Show others and affiliations
2013 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 21, 5398-5407 p.Article in journal (Refereed) Published
Abstract [en]

Recombinant Ca2+-ATPase was expressed in Saccharomycescerevisiae with a biotin-acceptor domain linked to its C-terminus by a thrombin cleavage site. We obtained 200g of similar to 70% pure recombinant sarcoendoplasmic reticulum Ca2+-ATPase isoform1a (SERCA1a) from a 6-L yeast culture. The catalytic cycle of SERCA1a was followed in real time using rapid scan FTIR spectroscopy. Different intermediate states (Ca(2)E1P and Ca(2)E2P) of the recombinant protein were accumulated using different buffer compositions. The difference spectra of their formation from Ca(2)E1 had the same spectral features as those from the native rabbit SERCA1a. The enzyme-specific activity for the active enzyme fraction in both samples was also similar. The results show that the recombinant protein obtained from the yeast-based expression system has similar structural and dynamic properties as native rabbit SERCA1a. It is now possible to apply this expression system together with IR spectroscopy to the investigation of the role of individual amino acids.

Place, publisher, year, edition, pages
2013. Vol. 280, no 21, 5398-5407 p.
Keyword [en]
Ca2+-ATPase, IR spectroscopy, phosphoenzyme, recombinant protein, Saccharomycescerevisiae
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-98723DOI: 10.1111/febs.12131ISI: 000328622200015OAI: oai:DiVA.org:su-98723DiVA: diva2:685177
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council
Note

AuthorCount:5;

Available from: 2014-01-09 Created: 2014-01-09 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Infrared spectroscopy: a tool for protein characterization
Open this publication in new window or tab >>Infrared spectroscopy: a tool for protein characterization
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Infrared (IR) spectroscopy, which belongs to vibrational spectroscopy, detects the vibrations of molecules, for example, proteins. The absorption of the peptide group gives rise to 9 characteristic bands in the infrared region, named A, B, I-VII, with a decreasing energy or wavenumber (cm-1). Among the 9 bands, amide I, which is mainly caused by C=O stretching vibration, is most sensitive to backbone structure and environment, and therefore can be used for structural analysis. In this thesis, a membrane protein sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) and a self-assembling peptide was studied with IR spectroscopy.  

In the first two papers, IR spectroscopy was used to assess the quality of a recombinant SERCA1a. A yeast-based expression system was applied to express recombinant SERCA1a, and the reaction cycle as well as the structure was analysed with IR spectroscopy. Different reaction intermediates were accumulated under different buffer conditions upon the release of ATP. The results showed that the recombinant protein shared similar IR features compared to the native protein. However, two SERCA1a preparations showed a difference around 1640 cm-1 in the amide I region. Using curve fitting, the band was assigned to β structure, and further investigation indicated that the difference in this region originates from protein aggregation. In the third paper, a co-fitting approach was tested and showed to be a more reliable method for structural analysis, and it can be applied in the biological IR spectroscopy. In the fourth paper, a peptide was computational designed and was predicted to self-assemble to amyloid fibrils, the formation of the fibril was confirmed with both electron microscopy and X-ray diffraction. IR spectroscopy was used to analyze further the structural details and the results support our structural predication. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2016
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-128761 (URN)978-91-7649-407-3 (ISBN)
Public defence
2016-05-27, Magnéli Hall, Arrhenius Laboratory, Svante arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2016-05-02 Created: 2016-04-04 Last updated: 2017-02-20Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Li, ChengeBarth, Andreas
By organisation
Department of Biochemistry and Biophysics
In the same journal
The FEBS Journal
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 21 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