In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties
The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein <i>Aequorea victoria</i> enhanced green fluorescent protein...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-02-01
|
| Series: | Molecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1420-3049/28/4/1901 |
| _version_ | 1797619031008608256 |
|---|---|
| author | Victor Marchenkov Tanya Ivashina Natalia Marchenko Natalya Ryabova Olga Selivanova Alexander Timchenko Hiroshi Kihara Vladimir Ksenzenko Gennady Semisotnov |
| author_facet | Victor Marchenkov Tanya Ivashina Natalia Marchenko Natalya Ryabova Olga Selivanova Alexander Timchenko Hiroshi Kihara Vladimir Ksenzenko Gennady Semisotnov |
| author_sort | Victor Marchenkov |
| collection | DOAJ |
| description | The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein <i>Aequorea victoria</i> enhanced green fluorescent protein (EGFP) or <i>Gaussia princeps</i> luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL<sub>14</sub>-EGFP is evenly distributed within normally divided <i>E. coli</i> cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL<sub>14</sub> which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL<sub>14</sub> containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES. |
| first_indexed | 2024-03-11T08:21:10Z |
| format | Article |
| id | doaj.art-96c2218b9bc249968a2010a1f74b92db |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-11T08:21:10Z |
| publishDate | 2023-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-96c2218b9bc249968a2010a1f74b92db2023-11-16T22:24:49ZengMDPI AGMolecules1420-30492023-02-01284190110.3390/molecules28041901In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional PropertiesVictor Marchenkov0Tanya Ivashina1Natalia Marchenko2Natalya Ryabova3Olga Selivanova4Alexander Timchenko5Hiroshi Kihara6Vladimir Ksenzenko7Gennady Semisotnov8Institute of Protein Research, Russian Academy of Sciences, 4 Institutskaya St., 142290 Pushchino, RussiaSkryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 5 Prospect Nauki, 142290 Pushchino, RussiaInstitute of Protein Research, Russian Academy of Sciences, 4 Institutskaya St., 142290 Pushchino, RussiaInstitute of Protein Research, Russian Academy of Sciences, 4 Institutskaya St., 142290 Pushchino, RussiaInstitute of Protein Research, Russian Academy of Sciences, 4 Institutskaya St., 142290 Pushchino, RussiaInstitute of Protein Research, Russian Academy of Sciences, 4 Institutskaya St., 142290 Pushchino, RussiaDepartment of Physics, Kansai Medical University, Shin-Machi 2-5-1, Hirakata 573-1010, JapanInstitute of Protein Research, Russian Academy of Sciences, 4 Institutskaya St., 142290 Pushchino, RussiaInstitute of Protein Research, Russian Academy of Sciences, 4 Institutskaya St., 142290 Pushchino, RussiaThe incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein <i>Aequorea victoria</i> enhanced green fluorescent protein (EGFP) or <i>Gaussia princeps</i> luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL<sub>14</sub>-EGFP is evenly distributed within normally divided <i>E. coli</i> cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL<sub>14</sub> which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL<sub>14</sub> containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.https://www.mdpi.com/1420-3049/28/4/1901GroEL chaperoninphotoproteinsprotein–protein interactionsprotein oligomerizationcell division |
| spellingShingle | Victor Marchenkov Tanya Ivashina Natalia Marchenko Natalya Ryabova Olga Selivanova Alexander Timchenko Hiroshi Kihara Vladimir Ksenzenko Gennady Semisotnov In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties Molecules GroEL chaperonin photoproteins protein–protein interactions protein oligomerization cell division |
| title | In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties |
| title_full | In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties |
| title_fullStr | In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties |
| title_full_unstemmed | In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties |
| title_short | In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties |
| title_sort | in vivo incorporation of photoproteins into groel chaperonin retaining major structural and functional properties |
| topic | GroEL chaperonin photoproteins protein–protein interactions protein oligomerization cell division |
| url | https://www.mdpi.com/1420-3049/28/4/1901 |
| work_keys_str_mv | AT victormarchenkov invivoincorporationofphotoproteinsintogroelchaperoninretainingmajorstructuralandfunctionalproperties AT tanyaivashina invivoincorporationofphotoproteinsintogroelchaperoninretainingmajorstructuralandfunctionalproperties AT nataliamarchenko invivoincorporationofphotoproteinsintogroelchaperoninretainingmajorstructuralandfunctionalproperties AT natalyaryabova invivoincorporationofphotoproteinsintogroelchaperoninretainingmajorstructuralandfunctionalproperties AT olgaselivanova invivoincorporationofphotoproteinsintogroelchaperoninretainingmajorstructuralandfunctionalproperties AT alexandertimchenko invivoincorporationofphotoproteinsintogroelchaperoninretainingmajorstructuralandfunctionalproperties AT hiroshikihara invivoincorporationofphotoproteinsintogroelchaperoninretainingmajorstructuralandfunctionalproperties AT vladimirksenzenko invivoincorporationofphotoproteinsintogroelchaperoninretainingmajorstructuralandfunctionalproperties AT gennadysemisotnov invivoincorporationofphotoproteinsintogroelchaperoninretainingmajorstructuralandfunctionalproperties |