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Smirnova, Irina
Alternative names
Publications (4 of 4) Show all publications
Smirnova, I. A., Ädelroth, P. & Brzezinski, P. (2018). Extraction and liposome reconstitution of membrane proteins with their native lipids without the use of detergents. Scientific Reports, 8, Article ID 14950.
Open this publication in new window or tab >>Extraction and liposome reconstitution of membrane proteins with their native lipids without the use of detergents
2018 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 8, article id 14950Article in journal (Refereed) Published
Abstract [en]

Functional studies of membrane-bound channels, transporters or signal transducers require that the protein of interest resides in a membrane that separates two compartments. One approach that is commonly used to prepare these systems is to reconstitute the protein in liposomes. An intermediate step of this method is purification of the protein, which typically involves solubilization of the native membrane using detergent. The use of detergents often results in removal of lipids surrounding the protein, which may alter its structure and function. Here, we have employed a method for isolation of membrane proteins with a disc of their native lipids to develop an approach that allows transfer of the purified membrane protein to liposomes without the use of any detergents.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-161983 (URN)10.1038/s41598-018-33208-1 (DOI)000446577500027 ()30297885 (PubMedID)
Available from: 2018-11-20 Created: 2018-11-20 Last updated: 2022-09-15Bibliographically approved
von Ballmoos, C., Smirnova, I., Poiana, F., Gonska, N., Chang, H.-Y., Gennis, R. B., . . . Ädelroth, P. (2017). Dynamics of the K-B Proton Pathway in Cytochrome ba(3) from Thermus thermophilus. Israel Journal of Chemistry, 57(5), 424-436
Open this publication in new window or tab >>Dynamics of the K-B Proton Pathway in Cytochrome ba(3) from Thermus thermophilus
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2017 (English)In: Israel Journal of Chemistry, ISSN 0021-2148, Vol. 57, no 5, p. 424-436Article in journal (Refereed) Published
Abstract [en]

The ba(3) cytochrome c oxidase from Thermus thermophilus is a B-type oxygen-reducing heme-copper oxidase and a proton pump. It uses only one proton pathway for transfer of protons to the catalytic site, the K-B pathway. It was previously shown that the ba(3) oxidase has an overall similar reaction sequence to that in mitochondrial-like A-type oxidases. However, the timing of loading the pump site, and formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated variants in which two amino acids of the K-B proton pathway leading to the catalytic site were exchanged; Tyr-248 (located approximate to 23 angstrom below the active site towards the cytoplasm) in subunit I (Y248T) and Glu-15 (approximate to 26 angstrom below the active site, approximate to 16 angstrom from Tyr-248) in subunit II (E15(II)Q). Even though the overall catalytic turnover in these two variants is similar and very low (<1% of wildtype), the substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site. The results indicate that the Glu-15(II) is the only essentially crucial residue of the K-B pathway, but that the Tyr-248 also plays a distinct role in defining an internal proton donor and controlling the dynamics of proton transfer to the pump site and the catalytic site.

Keywords
heme-copper oxidases, cytochrome c oxidase, proton transfer, electron transfer, membrane protein, respiration, redox reaction, metalloprotein, cytochrome aa(3), cytochrome cbb(3)
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-144708 (URN)10.1002/ijch.201600136 (DOI)000401329000009 ()
Available from: 2017-07-21 Created: 2017-07-21 Last updated: 2022-02-28Bibliographically approved
Smirnova, I. A., Sjöstrand, D., Li, F., Björck, M., Schäfer, J., Östbye, H., . . . Brzezinski, P. (2016). Isolation of yeast complex IV in native lipid nanodiscs. Biochimica et Biophysica Acta - Biomembranes, 1858(12), 2984-2992
Open this publication in new window or tab >>Isolation of yeast complex IV in native lipid nanodiscs
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2016 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, no 12, p. 2984-2992Article in journal (Refereed) Published
Abstract [en]

We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11 nm x 14 nm. Based on this size we estimated that each CytcO was surrounded by similar to 100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though CytcO forms a supercomplex with cytochrome bc(1) in the mitochondria! membrane, cyt.bc(1) was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated CytcO. The native nanodiscs displayed an O-2-reduction activity of similar to 130 electrons CytcO(-1) s(-1) and the kinetics of the reaction of the fully reduced CytcO with 02 was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the CytcO catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.

Keywords
Bioenergetics, Proton transfer, Membrane protein, Energy conservation
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-136722 (URN)10.1016/j.bbamem.2016.09.004 (DOI)000388048600004 ()27620332 (PubMedID)
Available from: 2016-12-19 Created: 2016-12-14 Last updated: 2022-02-28Bibliographically approved
Smirnova, I., Chang, H.-Y., von Ballmoos, C., Adelroth, P., Gennis, R. B. & Brzezinski, P. (2013). Single Mutations That Redirect Internal Proton Transfer in the ba(3) Oxidase from Thermus thermophilus. Biochemistry, 52(40), 7022-7030
Open this publication in new window or tab >>Single Mutations That Redirect Internal Proton Transfer in the ba(3) Oxidase from Thermus thermophilus
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2013 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 40, p. 7022-7030Article in journal (Refereed) Published
Abstract [en]

The ba(3)-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound proton pump. Results from earlier studies have shown that with the aa(3)-type oxidases proton uptake to the catalytic site and pump site occurs simultaneously. However, with ba(3) oxidase the pump site is loaded before proton transfer to the catalytic site because the proton transfer to the latter is slower than that with the aa(3) oxidases. In addition, the timing of formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated two mutant ba(3) CytcOs in which residues of the proton pathway leading to the catalytic site as well as the pump site were exchanged, Thr312Val and Tyr244Phe. Even though ba(3) CytcO uses only a single proton pathway for transfer of the substrate and pumped protons, the amino-acid residue substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site and the pump site. The results indicate that the rates of these reactions can be modified independently by replacement of single residues within the proton pathway. Furthermore, the data suggest that the Thr312Val and Tyr244Phe mutations interfere with a structural rearrangement in the proton pathway that is rate limiting for proton transfer to the catalytic site.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2013
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-97038 (URN)10.1021/bi4008726 (DOI)000326355500010 ()
Note

AuthorCount:6;

Funding Agencies:

Swedish Research Council;  National Institutes of Health HL 16101;

Stockholm University  

Available from: 2013-12-03 Created: 2013-12-02 Last updated: 2022-02-24Bibliographically approved
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