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
Use of Creatine Kinase To Induce Multistep Reactions in Infrared Spectroscopic Experiments
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2013 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 48, p. 14967-14972Article in journal (Refereed) Published
Abstract [en]

An extension of current approaches to trigger enzymatic reactions in reaction-induced infrared difference spectroscopy experiments is described. A common procedure is to add a compound that induces a reaction in the protein of interest. To be able to induce multistep reactions, we explored here the use of creatine kinase (CK) for the study of phosphate transfer mechanisms. The enzymatic reaction of CK could be followed using bands at 1614 and 979 cm(-1) for creatine phosphate consumption, at 944 cm(-1) for ADP consumption, and at 1243, 992, and 917 cm(-1) for ATP formation. The potential of CK to induce multistep reactions in infrared spectroscopic experiments was demonstrated using the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) as the protein of interest. ADP binding to the ATPase was triggered by photolytic release of ADP from P-3-1-(2-nitro)phenylethyl ADP (caged ADP). CK added in small amounts converted the released ADP to ATP on the time scale of minutes. This phosphorylated the ATPase and led to the formation of the first phosphoenzyme intermediate Ca(2)E1P. Thus a difference spectrum could be obtained that reflected the reaction from the ADP ATPase complex to the first phosphoenzyme intermediate. Comparison with a phosphorylation spectrum obtained when the initial state was the ATP ATPase complex revealed the contribution of ATP's gamma-phosphate to the conformational change of the ATPase upon nucleotide binding: gamma-phosphate binding modifies the structure of a beta-sheet, likely in the phosphorylation domain, and shifts its spectral position from similar to 1640 to similar to 1630 cm(-1). Upon phosphorylation of the ATPase, the beta-sheet relaxes back to a structure that is intermediate between that adopted in the ADP bound state and that in the ATP bound state.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2013. Vol. 117, no 48, p. 14967-14972
National Category
Physical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-98722DOI: 10.1021/jp409599pISI: 000328101100009OAI: oai:DiVA.org:su-98722DiVA, id: diva2:685180
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council
Note

AuthorCount:2;

Available from: 2014-01-09 Created: 2014-01-09 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Infrared spectroscopic studies: from small molecules to large
Open this publication in new window or tab >>Infrared spectroscopic studies: from small molecules to large
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Infrared light (IR) was first discovered by Friedrich Wilhelm Herschel in 1800. However, until 1940’s, molecular IR studies involved only water and small organic molecules, because of the long measurement times. Development Fourier transform infrared spectroscopy (FTIR) has minimized the time required to obtain data, making it possible to investigate bigger biological systems, e.g. proteins and nucleic acids.This thesis concentrates on the applications of different IR spectroscopic techniques to a variety of biological systems and development of new approaches to study complicated biological events.

The first paper in this work concerns using so-called caged compounds to study the aggregation of Alzheimer’s Aβ-peptide which is linked to the formation of neurotoxic fibrils in the brain. By adding caged-sulfate to the Aβ samples we were able to change the pH of the sample, while recording IR data and study fibril formation in a time-resolved manner. Then we used caged–ADP to study the production of ATP and creatine, mediated by creatine kinase (CK). Using CK as a helper enzyme we studied the effects of the phosphate binding on the secondary structure of SR Ca2+ATPse and determined the structural differences between two similar states Ca2E1ADP and Ca2E1ATP.

In the second part of the thesis we used ATR-FTIR spectroscopy and a specially designed dialysis setup, to develop a general method to detect ligand binding events by observing the IR absorbance changes in the water hydration shell around the molecules. The same method was used to determine the binding of DNA to the transcription factors of the E2F family. E2F proteins play main part in the gene regulatory networks that control cell development. However how they recognize their DNA-binding sites and the mechanism of binding is not well understood. By using ATR-FTIR, we observed the changes in the secondary structure of the proteins, as well as the distortions to the DNA upon E2F-DNA complex formation.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. p. 59
Keywords
Infrared spectroscopy, transcription factors, DNA, creatine kinase, CaATPase, water, ligand binding
National Category
Biophysics
Research subject
Biopharmaceutics
Identifiers
urn:nbn:se:su:diva-101077 (URN)978-91-7447-876-1 (ISBN)
Public defence
2014-03-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2014-03-06 Created: 2014-02-24 Last updated: 2014-02-26Bibliographically approved

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full text

Search in DiVA

By author/editor
Eremina, NadejdaBarth, Andreas
By organisation
Department of Biochemistry and Biophysics
In the same journal
Journal of Physical Chemistry B
Physical Chemistry

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 32 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