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Infrared spectroscopic studies: from small molecules to large
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Andreas Barth)
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. , 59 p.
Keyword [en]
Infrared spectroscopy, transcription factors, DNA, creatine kinase, CaATPase, water, ligand binding
National Category
Biophysics
Research subject
Biopharmaceutics
Identifiers
URN: urn:nbn:se:su:diva-101077ISBN: 978-91-7447-876-1 (print)OAI: oai:DiVA.org:su-101077DiVA: diva2:698703
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
List of papers
1. Formation of Two Different Types of Oligomers in the Early Phase of pH-Induced Aggregation of the Alzheimer A beta(12-28) Peptide
Open this publication in new window or tab >>Formation of Two Different Types of Oligomers in the Early Phase of pH-Induced Aggregation of the Alzheimer A beta(12-28) Peptide
2012 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 116, no 41, 12389-12397 p.Article in journal (Refereed) Published
Abstract [en]

The early phase in the aggregation process of the Alzheimer's peptide A beta(12-28) with both protected and unprotected ends was studied by time-resolved infrared spectroscopy and circular dichroism spectroscopy. Aggregation in the time-resolved experiments was initiated by a rapid pH drop caused by the photolysis of 1-(2-nitrophenyl)ethyl sulfate (caged sulfate). The infrared spectra indicate two different types of aggregates from both versions of the A beta(12-28) peptide. One type has small and/or twisted beta sheets with a beta-sheet band at 1627 cm(-1), They form fast (within 60 ms), presumably from initial aggregates, and their spectral signature is consistent with a beta-barrel structure. The other type arises relatively slowly from unstructured monomers on the seconds-to-minutes time scale and forms at lower pH than the first type. These beta sheets are antiparallel, planar, and large and show an absorption band at 1622 cm(-1) that shifts to 1617 cm(-1) in 12 min with most of the shift occurring in 10 s.

National Category
Physical Chemistry
Identifiers
urn:nbn:se:su:diva-83008 (URN)10.1021/jp305015g (DOI)000309902400002 ()
Note

AuthorCount:3;

Available from: 2012-12-05 Created: 2012-12-03 Last updated: 2017-12-07Bibliographically approved
2. Use of Creatine Kinase To Induce Multistep Reactions in Infrared Spectroscopic Experiments
Open this publication in new window or tab >>Use of Creatine Kinase To Induce Multistep Reactions in Infrared Spectroscopic Experiments
2013 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 48, 14967-14972 p.Article 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
National Category
Physical Chemistry
Identifiers
urn:nbn:se:su:diva-98722 (URN)10.1021/jp409599p (DOI)000328101100009 ()
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
3. Detection of Ligand Binding to Proteins through Observation of Hydration Water
Open this publication in new window or tab >>Detection of Ligand Binding to Proteins through Observation of Hydration Water
2012 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 116, no 48, 13968-13974 p.Article in journal (Refereed) Published
Abstract [en]

Drug development is impeded by the need to design for each drug target a test that detects the binding of drug candidate molecules to the target protein. Therefore, a general method to detect ligand binding is highly desirable. Here, we present an observation toward developing such a method, which is based on monitoring a change in water absorption by infrared spectroscopy. Infrared spectroscopy has high sensitivity for water, and changes in its hydrogen bond pattern can be observed. We studied absorption changes of water upon the addition of phosphenolpyruvate or Mg2+ to pyruvate kinase. In each case, there is a decrease in the absorption of water in the 3000-3100 cm(-1) region on the low wavenumber side of the OH stretching vibration when a ligand binds to the protein. Our results suggest that the weaker water absorption is due to the release of protein-bound water into bulk water during ligand binding. This observation has high potential for drug development as well as for basic research because it can lead to a general method for detecting molecular association events that (i) is label-free, (ii) works with both binding partners being in aqueous solution, and (iii) is based on a universal process that takes place in all binding events.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-84809 (URN)10.1021/jp307560r (DOI)000311921700003 ()
Note

AuthorCount:3;

Available from: 2013-01-02 Created: 2013-01-02 Last updated: 2017-12-06Bibliographically approved
4. Interaction between Transcription Factors of E2F family and DNA Studied with Infrared Spectroscopy
Open this publication in new window or tab >>Interaction between Transcription Factors of E2F family and DNA Studied with Infrared Spectroscopy
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The E2F transcription factors are crucial for the regulation of genes required for proper progression through the cell cycle. Since E2Fs can initiate both cell proliferation and cell death, it is not surprising that these proteins are the subjects of extensive studies. In this work we characterize the formation of E2F1- and E2F8-DNA complexes with Fourier transform infrared spectroscopy. We demonstrate the changes in the secondary structure of the E2F1 and E2F8, in particular the disappearance of regular α-helices. We also show the perturbation to the DNA double helix and characterize the interactions between the amino acids in the proteins and the bases in the DNA.

National Category
Biophysics
Identifiers
urn:nbn:se:su:diva-101086 (URN)
Available from: 2014-02-25 Created: 2014-02-25 Last updated: 2014-02-25

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