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The choreography of protein vibrations: Improved methods of observing and simulating the infrared absorption of proteins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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 [en]
Infrared spectroscopy, FTIR, protein, atpase, amyloid, caged compound, amide I, transition dipole coupling, exciton theory, simulation
National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-60415ISBN: 978-91-7447-322-3 (print)OAI: oai:DiVA.org:su-60415DiVA: diva2:435158
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
List of papers
1. Use of Helper Enzymes for ADP Removal in Infrared Spectroscopic Experiments: Application to Ca2+-ATPase
Open this publication in new window or tab >>Use of Helper Enzymes for ADP Removal in Infrared Spectroscopic Experiments: Application to Ca2+-ATPase
2005 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 88, no 5, 3615-3624 p.Article in journal (Refereed) Published
Abstract [en]

Adenylate kinase (AdK) and apyrase were employed as helper enzymes to remove ADP in infrared spectroscopic experiments that study the sarcoplasmic reticulum Ca2+-ATPase. The infrared absorbance changes of their enzymatic reactions were characterized and used to monitor enzyme activity. AdK transforms ADP to ATP and AMP, whereas apyrase consumes ATP and ADP to generate AMP and inorganic phosphate. The benefits of using them as helper enzymes are severalfold: i), both remove ADP generated after ATP hydrolysis by ATPase, which enables repeat of ATP-release experiments several times with the same sample without interference by ADP; ii), AdK helps maintain the presence of ATP for a longer time by regenerating 50% of the initial ATP; iii), apyrase generates free Pi, which can help stabilize the ADP-insensitive phosphoenzyme (E2P); and iv), apyrase can be used to monitor ADP dissociation from transient enzyme intermediates with relatively high affinity to ADP, as shown here for ADP dissociation from the ADP-sensitive phosphoenzyme intermediate (Ca2E1P). The respective infrared spectra indicate that ADP dissociation relaxes the closed conformation immediately after phosphorylation partially back toward the open conformation of Ca2E1 but does not trigger the transition to E2P. The helper enzyme approach can be extended to study other nucleotide-dependent proteins.

National Category
Natural Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-61610 (URN)10.1529/biophysj.104.055368 (DOI)
Available from: 2011-08-23 Created: 2011-08-23 Last updated: 2017-12-08Bibliographically approved
2. Toward a general method to observe the phosphate groups of phosphoenzymes with infrared spectroscopy
Open this publication in new window or tab >>Toward a general method to observe the phosphate groups of phosphoenzymes with infrared spectroscopy
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.

National Category
Natural Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-61609 (URN)10.1529/biophysj.106.084442 (DOI)
Available from: 2011-08-23 Created: 2011-08-23 Last updated: 2017-12-08Bibliographically approved
3. Proton paths in the sarcoplasmic reticulum Ca2+-ATPase
Open this publication in new window or tab >>Proton paths in the sarcoplasmic reticulum Ca2+-ATPase
2007 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1767, no 11, 1310-1318 p.Article in journal (Refereed) Published
Abstract [en]

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) pumps Ca(2+) and countertransport protons. Proton pathways in the Ca(2+) bound and Ca(2+)-free states are suggested based on an analysis of crystal structures to which water molecules were added. The pathways are indicated by chains of water molecules that interact favorably with the protein. In the Ca(2+) bound state Ca(2)E1, one of the proposed Ca(2+) entry paths is suggested to operate additionally or alternatively as proton pathway. In analogs of the ADP-insensitive phosphoenzyme E2P and in the Ca(2+)-free state E2, the proton path leads between transmembrane helices M5 to M8 from the lumenal side of the protein to the Ca(2+) binding residues Glu-771, Asp-800 and Glu-908. The proton path is different from suggested Ca(2+) dissociation pathways. We suggest that separate proton and Ca(2+) pathways enable rapid (partial) neutralization of the empty cation binding sites. For this reason, transient protonation of empty cation binding sites and separate pathways for different ions are advantageous for P-type ATPases in general.

Keyword
SERCA1a, Ca2+ pump, Na, K-ATPase, Proton countertransport, Proton pathway
National Category
Natural Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-19427 (URN)000251406600005 ()17904096 (PubMedID)
Available from: 2009-01-14 Created: 2009-01-14 Last updated: 2017-12-13Bibliographically approved
4. Simulation of the Amide I Absorption of Stacked β-Sheets
Open this publication in new window or tab >>Simulation of the Amide I Absorption of Stacked β-Sheets
2011 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 115, no 4, 749-757 p.Article in journal (Refereed) Published
Abstract [en]

Aggregated β-sheet structures are associated with amyloid and prion diseases. Techniques capable of revealing detailed structural and dynamical information on β-sheet structure are thus of great biomedical and biophysical interest. In this work, the infrared (IR) amide I spectral characteristics of stacked β-sheets were modeled using the transition dipole coupling model. For a test set of β-sheet stacks, the simulated amide I spectrum was analyzed with respect to the following parameters; intersheet distance, relative rotation of the sheets with respect to each other and the effect of number of sheets stacked. The amide I maximum shifts about 5 cm(-1) to higher wavenumbers when the intersheet distance between two identical β-sheets decreases from 20 to 5 Å. Rotation around the normal of one of the sheets relative to the other results in maximum intersheet coupling near 0° and 180°. Upon of rotation from 0° to 90° at an intersheet distance of 9 Å, the amide I maximum shifts about 3 cm(-1). Tilting of one of the sheets by 30° from the normal results in a shift of the amide I maximum by less than 1 cm(-1). When stacking several β-sheets along the normal, the amide I maximum shifts to higher wavenumbers with increasing stack size. The amide I maximum shifts about 6 cm(-1) when stacking four sheets with an intersheet distance of 9 Å. The study provides an aid in the interpretation of the IR amide I region for experiments involving β-sheets and creates awareness of the many effects that determine the spectrum of β-sheet structures.

Keyword
IR amide I, β-sheets, β-sheet structures
National Category
Natural Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-52568 (URN)10.1021/jp109918c (DOI)000286639500019 ()21174476 (PubMedID)
Available from: 2011-01-17 Created: 2011-01-17 Last updated: 2017-12-11Bibliographically approved
5. Optimization of Model Parameters for Describing the Amide I Spectrum of a Large Set of Proteins
Open this publication in new window or tab >>Optimization of Model Parameters for Describing the Amide I Spectrum of a Large Set of Proteins
2012 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 116, no 16, 4831-4842 p.Article in journal (Refereed) Published
Abstract [en]

A new simulation protocol for the prediction of the infrared absorption of the amide I vibration of proteins was developed. The method incorporates known effects on the intrinsic frequencies (backbone conformation, interpeptide and peptide-solvent hydrogen bonding) and couplings (nearest neighbor coupling, transition dipole coupling) of amide I oscillators in a parametrized manner. Model parameters for the simulation of amide I spectra were determined through fitting and optimization of simulated spectra to experimentally measured infrared spectra of 44 proteins that represent maximum structural variation in terms of different folds and secondary structure contents. Prediction of protein spectra using the optimized parameters resulted in good agreement with experimental spectra and in a considerable improvement compared to a description involving only transition dipole coupling.

National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-80301 (URN)10.1021/jp301095v (DOI)000303173800012 ()
Note

AuthorCount:3;

Reprinted with permission from http://pubs.acs.org/doi/abs/10.1021%2Fjp301095v. Copyright 2012 American Chemical Society.

Available from: 2012-09-19 Created: 2012-09-17 Last updated: 2017-12-07Bibliographically approved

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