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A dialysis accessory for attenuated total reflection infrared spectroscopy
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2006 (English)In: Spectroscopy, ISSN 0712-4813, Vol. 20, no 3, 89-94 p.Article in journal (Refereed) Published
Abstract [en]

A dialysis accessory for attenuated total reflection (ATR) infrared spectroscopy is described together with an evaluation based on known systems with well-studied infrared spectra, such as chemical oxidation and reduction of cytochrome c and substrate binding to the Ca2+-ATPase. Changes in the infrared spectra of the two proteins are successfully monitored with the dialysis accessory. The accessory was developed in our laboratory for the diamond 9-reflections SensIR ATR unit. It can be used to study absorbance changes of macromolecules which are induced by low molecular weight compounds, for example the binding of substrates, inhibitors or ions to macromolecules as well as effects of pH, ionic strength or denaturants on the structure of macromolecules. The dialysis accessory confines the macromolecule of interest to a sample compartment created between the ATR crystal and the dialysis membrane. On the other side of the dialysis membrane, a reservoir for the sample medium is created. In this way the low molecular weight compound of interest can exchange freely between the reservoir and the sample compartment via the dialysis membrane. This provides a flexible way to change sample conditions for the macromolecule of interest, allowing for example initiation of ligand binding.

Place, publisher, year, edition, pages
2006. Vol. 20, no 3, 89-94 p.
Keyword [en]
Dialysis, attenuated total reflection, ATR, vibrational spectroscopy, FTIR, ligand binding, protein
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-56930OAI: oai:DiVA.org:su-56930DiVA: diva2:413959
Available from: 2011-05-01 Created: 2011-05-01 Last updated: 2011-05-01Bibliographically approved
In thesis
1. Infrared studies: Method development and binding of ligands to pyruvate kinase
Open this publication in new window or tab >>Infrared studies: Method development and binding of ligands to pyruvate kinase
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Infrared spectroscopy is a valuable technique for the study of ligand induce change in biomolecules. Our development of a dialysis accessory to attenuated total reflection infrared spectroscopy makes this technique more universal for ligand binding studies. We use this method to understand the binding of phosphoenolpyruvate (PEP) and Mg2+ to pyruvate kinase (PK), where conformational changes of PK were revealed upon binding of PEP and Mg2+. To investigate the effect of the protein environment on the bound PEP, we used labeled PEP, which helped assign and evaluate the infrared absorption bands. The effects of different divalent and monovalent ions on PEP binding to PK were also studied. We could demonstrate that the β-sheets were perturbed differently with Na+ as compared to the other monovalent ions. The pattern of structural changes does not correlate with the activity profiles of the monovalent ions. Thus, it seems unlikely that the ion effects on activity are due to the ion effects on the structure of the PEP:PK complex. Comparing different divalent ions, a particularly large conformational change and a more homogeneous binding mode was observed with Mn2+ and attributed to a more closed conformation of the complex. The allosteric effect of fructose 1, 6 bisphosphate (FBP) on PEP binding to PK in presence of various ions (Mg2+, Mn2+, K+, Na+) was studied. The experiments indicated that the conformational change of PEP binding to PK:Mg2+:K+ in the presence of FBP was about twice as large as in its absence, which is tentatively ascribed to a higher occupancy of the closed state of PK. The affinity for PEP increased in presence of Mg2+ and K+. No allosteric effects were observed with the other ion combinations Mn2+/K+ and Mg2+/Na+. A method of ligand binding by observing a change in water absorption was developed and established with four different proteins. The results suggest that the decrease of water absorption is due to the release of bound water into the bulk during the ligand binding process, which can be a used as label-free indicator of ligand-protein binding.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. 7 p.
National Category
Natural Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-56754 (URN)978-91-7447-297-4 (ISBN)
Public defence
2011-05-31, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Accepted. Paper 4: Manuscript. Paper 5: Manuscript.Available from: 2011-05-09 Created: 2011-04-26 Last updated: 2012-01-18Bibliographically approved

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