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
Infrared studies: Method development and binding of ligands to pyruvate kinase
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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: urn:nbn:se:su:diva-56754ISBN: 978-91-7447-297-4 (print)OAI: oai:DiVA.org:su-56754DiVA: diva2:412890
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
List of papers
1. A dialysis accessory for attenuated total reflection infrared spectroscopy
Open this publication in new window or tab >>A dialysis accessory for attenuated total reflection infrared spectroscopy
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.

Keyword
Dialysis, attenuated total reflection, ATR, vibrational spectroscopy, FTIR, ligand binding, protein
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-56930 (URN)
Available from: 2011-05-01 Created: 2011-05-01 Last updated: 2011-05-01Bibliographically approved
2. Phosphoenolpyruvate and Mg2+ binding to pyruvate kinase monitored by infrared spectroscopy
Open this publication in new window or tab >>Phosphoenolpyruvate and Mg2+ binding to pyruvate kinase monitored by infrared spectroscopy
2010 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 98, no 9, 1931-1940 p.Article in journal (Refereed) Published
Abstract [en]

Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg2+ binding were studied by attenuated total reflection Fourier transform infrared spectroscopy in combination with a dialysis accessory. The experiments indicated a largely preserved secondary structure upon PEP and Mg2+ binding but also revealed small backbone conformational changes of PK involving all types of secondary structure. To assess the effect of the protein environment on the bound PEP, we assigned and evaluated the infrared absorption bands of bound PEP. These were identified using 2,3-C-13(2)-labeled PEP. We obtained the following assignments: 1589 cm(-1) (antisymmetric carboxylate stretching vibration); 1415 cm(-1) (symmetric carboxylate stretching vibration); 1214 cm(-1) (C-O stretching vibration); 1124 and 1110 cm(-1) (asymmetric PO32- stretching vibrations); and 967 cm(-1) (symmetric PO32- stretching vibration). The corresponding band positions in solution are 1567, 1407, 1229, 1107, and 974 cm-1. The differences for bound and free PEP indicate specific interactions between ligand and protein. Quantification of the interactions with the phosphate group indicated that the enzyme environment has little influence on the P-O bond strengths, and that the bridging P-O bond, which is broken in the catalytic reaction, is weakened by <3%. Thus, there is only little distortion toward a dissociative transition state of the phosphate transfer reaction when PEP binds to PK. Therefore, our results are in line with an associative transition state. Carboxylate absorption bands indicated a maximal shortening of the length of the shorter C-O bond by 1.3 pm. PEP bound to PK in the presence of the monovalent ion Na+ exhibited the same band positions as in the presence of K+, indicating very similar interaction strengths between ligand and protein in both cases.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-49234 (URN)10.1016/j.bpj.2009.12.4335 (DOI)000277377300027 ()
Note
authorCount :2Available from: 2010-12-13 Created: 2010-12-13 Last updated: 2017-12-11Bibliographically approved
3. Effects of ions on ligand binding to pyruvate kinase: Mapping the binding site by infrared spectroscopy
Open this publication in new window or tab >>Effects of ions on ligand binding to pyruvate kinase: Mapping the binding site by infrared spectroscopy
(English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207Article in journal (Refereed) Accepted
National Category
Natural Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-56749 (URN)
Available from: 2011-04-26 Created: 2011-04-26 Last updated: 2017-12-11Bibliographically approved
4. The Allosteric Effect of Fructose Bisphosphate on Muscle Pyruvate Kinase Studied by Infrared Spectroscopy
Open this publication in new window or tab >>The Allosteric Effect of Fructose Bisphosphate on Muscle Pyruvate Kinase Studied by Infrared Spectroscopy
2011 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 115, no 39, 11501-11505 p.Article in journal (Refereed) Published
Abstract [en]

Pyruvate kinase exhibits allosteric properties. The allosteric effect of fructose 1,6-bisphosphate (FBP) on phosphoenolpyruvate (PEP) binding to rabbit muscle pyruvate kinase (PK) in the presence of various ions (mg(2+), mn(2+), Na(+)) was studied by attenuated total reflection infrared spectroscopy in combination with a dialysis accessory. The experiments indicated that FBP binding causes conformational changes of PK that are of the same order of magnitude as those of PEP binding. The conformational change of PEP binding to PK/Mg(2+)/K(+) in the presence of FBP was about twice as large as in its absence, which is tentatively ascribed to a higher occupancy the closed state. The affinity for PEP increased in the presence of Mg(2+) and K(+). No such effects were observed with the other ion combinations Mn(2+)/K(+) and Mg(2+)/Na(+) or in D(2)O (with Mg(2+)/K(+)), and therefore we did not detect an allosteric effect on PEP binding under these conditions.

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-69877 (URN)10.1021/jp206272x (DOI)000295245400022 ()
Note
authorCount :2Available from: 2012-01-18 Created: 2012-01-15 Last updated: 2017-12-08Bibliographically approved
5. Ligand binding detected by change in water absorption by infrared spectroscopy
Open this publication in new window or tab >>Ligand binding detected by change in water absorption by infrared spectroscopy
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-56752 (URN)
Available from: 2011-04-26 Created: 2011-04-26 Last updated: 2011-05-01Bibliographically approved

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Kumar, Saroj
By organisation
Department of Biochemistry and Biophysics
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 209 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