NMR Structure Determinations of Small Proteins Using only One Fractionally 20% <sup>13</sup>C- and Uniformly 100% <sup>15</sup>N-Labeled Sample
Uniformly <sup>13</sup>C- and <sup>15</sup>N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive an...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-02-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/26/3/747 |
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| author | Harri A. Heikkinen Sofia M. Backlund Hideo Iwaï |
| author_facet | Harri A. Heikkinen Sofia M. Backlund Hideo Iwaï |
| author_sort | Harri A. Heikkinen |
| collection | DOAJ |
| description | Uniformly <sup>13</sup>C- and <sup>15</sup>N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of <sup>13</sup>C-labeled glucose by a factor of five using a fractional 20% <sup>13</sup>C- and 100% <sup>15</sup>N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [<sup>13</sup>C, <sup>15</sup>N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional <sup>13</sup>C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% <sup>13</sup>C, 100% <sup>15</sup>N]-labeled sample for small proteins (<15 kDa) could also eliminate redundant sample preparations of 100% <sup>15</sup>N-labeled and uniformly 100% [<sup>13</sup>C, <sup>15</sup>N]-labeled samples of proteins. We determined the NMR structures of a small alpha-helical protein, the C domain of IgG-binding protein A from <i>Staphylococcus aureus</i> (SpaC), and a small beta-sheet protein, CBM64 module using [20% <sup>13</sup>C, 100% <sup>15</sup>N]-labeled sample and compared with the crystal structures and the NMR structures derived from the 100% [<sup>13</sup>C, <sup>15</sup>N]-labeled sample. Our results suggest that one [20% <sup>13</sup>C, 100% <sup>15</sup>N]-labeled sample of small proteins could be routinely used as an alternative to conventional 100% [<sup>13</sup>C, <sup>15</sup>N]-labeling for backbone resonance assignments, NMR structure determination, <sup>15</sup>N-relaxation analysis, and ligand–protein interaction. |
| first_indexed | 2024-03-09T06:15:05Z |
| format | Article |
| id | doaj.art-c1309ee16e734e1c8725bab623ee63e4 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-09T06:15:05Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-c1309ee16e734e1c8725bab623ee63e42023-12-03T11:54:13ZengMDPI AGMolecules1420-30492021-02-0126374710.3390/molecules26030747NMR Structure Determinations of Small Proteins Using only One Fractionally 20% <sup>13</sup>C- and Uniformly 100% <sup>15</sup>N-Labeled SampleHarri A. Heikkinen0Sofia M. Backlund1Hideo Iwaï2Institute of Biotechnology, University of Helsinki. P.O. Box 65, FIN-00014 Helsinki, FinlandInstitute of Biotechnology, University of Helsinki. P.O. Box 65, FIN-00014 Helsinki, FinlandInstitute of Biotechnology, University of Helsinki. P.O. Box 65, FIN-00014 Helsinki, FinlandUniformly <sup>13</sup>C- and <sup>15</sup>N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of <sup>13</sup>C-labeled glucose by a factor of five using a fractional 20% <sup>13</sup>C- and 100% <sup>15</sup>N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [<sup>13</sup>C, <sup>15</sup>N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional <sup>13</sup>C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% <sup>13</sup>C, 100% <sup>15</sup>N]-labeled sample for small proteins (<15 kDa) could also eliminate redundant sample preparations of 100% <sup>15</sup>N-labeled and uniformly 100% [<sup>13</sup>C, <sup>15</sup>N]-labeled samples of proteins. We determined the NMR structures of a small alpha-helical protein, the C domain of IgG-binding protein A from <i>Staphylococcus aureus</i> (SpaC), and a small beta-sheet protein, CBM64 module using [20% <sup>13</sup>C, 100% <sup>15</sup>N]-labeled sample and compared with the crystal structures and the NMR structures derived from the 100% [<sup>13</sup>C, <sup>15</sup>N]-labeled sample. Our results suggest that one [20% <sup>13</sup>C, 100% <sup>15</sup>N]-labeled sample of small proteins could be routinely used as an alternative to conventional 100% [<sup>13</sup>C, <sup>15</sup>N]-labeling for backbone resonance assignments, NMR structure determination, <sup>15</sup>N-relaxation analysis, and ligand–protein interaction.https://www.mdpi.com/1420-3049/26/3/747NMR structure determinationfractional <sup>13</sup>C-labelingbiosynthetically directed fractional <sup>13</sup>C-labelingNMRbiosynthetic pathwaysprotein structure |
| spellingShingle | Harri A. Heikkinen Sofia M. Backlund Hideo Iwaï NMR Structure Determinations of Small Proteins Using only One Fractionally 20% <sup>13</sup>C- and Uniformly 100% <sup>15</sup>N-Labeled Sample Molecules NMR structure determination fractional <sup>13</sup>C-labeling biosynthetically directed fractional <sup>13</sup>C-labeling NMR biosynthetic pathways protein structure |
| title | NMR Structure Determinations of Small Proteins Using only One Fractionally 20% <sup>13</sup>C- and Uniformly 100% <sup>15</sup>N-Labeled Sample |
| title_full | NMR Structure Determinations of Small Proteins Using only One Fractionally 20% <sup>13</sup>C- and Uniformly 100% <sup>15</sup>N-Labeled Sample |
| title_fullStr | NMR Structure Determinations of Small Proteins Using only One Fractionally 20% <sup>13</sup>C- and Uniformly 100% <sup>15</sup>N-Labeled Sample |
| title_full_unstemmed | NMR Structure Determinations of Small Proteins Using only One Fractionally 20% <sup>13</sup>C- and Uniformly 100% <sup>15</sup>N-Labeled Sample |
| title_short | NMR Structure Determinations of Small Proteins Using only One Fractionally 20% <sup>13</sup>C- and Uniformly 100% <sup>15</sup>N-Labeled Sample |
| title_sort | nmr structure determinations of small proteins using only one fractionally 20 sup 13 sup c and uniformly 100 sup 15 sup n labeled sample |
| topic | NMR structure determination fractional <sup>13</sup>C-labeling biosynthetically directed fractional <sup>13</sup>C-labeling NMR biosynthetic pathways protein structure |
| url | https://www.mdpi.com/1420-3049/26/3/747 |
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