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Toward a general method to observe the phosphate groups of phosphoenzymes with 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: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 91, no 6, 2282-2289 p.Article in journal (Refereed) Published
Abstract [en]

A general method to study the phosphate group of phosphoenzymes with infrared difference spectroscopy by helper enzyme-induced isotope exchange was developed. This allows the selective monitoring of the phosphate P-O vibrations in large proteins, which provides detailed information on several band parameters. Here, isotopic exchange was achieved at the oxygen atoms of the catalytically important phosphate group that transiently binds to the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a). [γ-18O3]ATP phosphorylated the ATPase, which produced phosphoenzyme that was initially isotopically labeled. The helper enzyme adenylate kinase regenerated the substrate ATP from ADP (added or generated upon ATP hydrolysis) with different isotopic composition than used initially. With time this produced the unlabeled phosphoenzyme. The method was tested on the ADP-insensitive phosphoenzyme state of the Ca2+-ATPase for which the vibrational frequencies of the phosphate group are known, and it was established that the helper enzyme is effective in mediating the isotope exchange process.

Place, publisher, year, edition, pages
2006. Vol. 91, no 6, 2282-2289 p.
National Category
Natural Sciences
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-61609DOI: 10.1529/biophysj.106.084442OAI: oai:DiVA.org:su-61609DiVA: diva2:436532
Available from: 2011-08-23 Created: 2011-08-23 Last updated: 2017-12-08Bibliographically approved
In thesis
1. The choreography of protein vibrations: Improved methods of observing and simulating the infrared absorption of proteins
Open this publication in new window or tab >>The choreography of protein vibrations: Improved methods of observing and simulating the infrared absorption of proteins
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The work presented in this thesis has striven toward improving the capability to study proteins using infrared (IR) spectroscopy. This includes development of new and improved experimental and theoretical methods to selectively observe and simulate protein vibrations.

A new experimental method of utilising adenylate kinase and apyrase as helper enzymes to alter the nucleotide composition and to perform isotope exchange in IR samples was developed. This method enhances the capability of IR spectroscopy by enabling increased duration of measurement time, making experiments more repeatable and allowing investigation of partial reactions and selected frequencies otherwise difficult to observe. The helper enzyme mediated isotope exchange allowed selective observation of the vibrations of the catalytically important phosphate group in a nucleotide dependent protein such as the sarcoplasmic reticulum Ca2+-ATPase. This important and representative member of P-type ATPases was further investigated in a different study, where a pathway for the protons countertransported in the Ca2+-ATPase reaction cycle was proposed based on theoretical considerations. The transport mechanism was suggested to involve separate pathways for the ions and the protons.

Simulation of the IR amide I band of proteins enables and supports structure-spectra correlations. The characteristic stacking of beta-sheets observed in amyloid structures was shown to induce a band shift in IR spectra based on simulations of the amide I band. The challenge of simulating protein spectra in aqueous medium was also addressed in a novel approach where optimisation of simulated spectra of a large set of protein structures to their corresponding experimental spectra was performed. Thereby, parameters describing the most important effects on the amide I band for proteins could be determined. The protein spectra predicted using the optimised parameters were found to be well in agreement with experiment.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. 88 p.
Keyword
Infrared spectroscopy, FTIR, protein, atpase, amyloid, caged compound, amide I, transition dipole coupling, exciton theory, simulation
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-60415 (URN)978-91-7447-322-3 (ISBN)
Public defence
2011-09-23, 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 5: Manuscript.

Available from: 2011-09-01 Created: 2011-08-16 Last updated: 2012-09-19Bibliographically approved

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