Engineering Encodable Lanthanide-Binding Tags (LBTs) into Loop Regions of Proteins
Lanthanide-binding tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography, and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1β (...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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American Chemical Society
2012
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| Online Access: | http://hdl.handle.net/1721.1/69924 https://orcid.org/0000-0002-5749-7869 |
| _version_ | 1826194788982980608 |
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| author | Barthelmes, Katja Reynolds, Anne M. Peisach, Ezra Jonker, Hendrik R. A. DeNunzio, Nicholas J. Allen, Karen N. Imperiali, Barbara Schwalbe, Harald |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Barthelmes, Katja Reynolds, Anne M. Peisach, Ezra Jonker, Hendrik R. A. DeNunzio, Nicholas J. Allen, Karen N. Imperiali, Barbara Schwalbe, Harald |
| author_sort | Barthelmes, Katja |
| collection | MIT |
| description | Lanthanide-binding tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography, and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1β (IL1β) and varied the length of the spacer between the LBT and the protein (denoted 1−3). Luminescence studies demonstrate that all nine constructs bind Tb3+ tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1β-S1 and IL1β-L3 constructs and for the remaining constructs by comparing the 1H−15N heteronuclear single-quantum coherence NMR spectra with that of the wild-type IL1β. Additionally, binding of LBT-loop IL1β proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loop constructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1β structure. The paramagnetic NMR spectra of loop-LBT mutant IL1β-R2 were assigned and the Δχ tensor components were calculated on the basis of RDCs and pseudocontact shifts. A structural model of the IL1β-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modeling. |
| first_indexed | 2024-09-23T10:02:08Z |
| format | Article |
| id | mit-1721.1/69924 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T10:02:08Z |
| publishDate | 2012 |
| publisher | American Chemical Society |
| record_format | dspace |
| spelling | mit-1721.1/699242022-09-30T18:26:39Z Engineering Encodable Lanthanide-Binding Tags (LBTs) into Loop Regions of Proteins Engineering Encodable Lanthanide-Binding Tags into Loop Regions of Proteins Barthelmes, Katja Reynolds, Anne M. Peisach, Ezra Jonker, Hendrik R. A. DeNunzio, Nicholas J. Allen, Karen N. Imperiali, Barbara Schwalbe, Harald Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Imperiali, Barbara Imperiali, Barbara Reynolds, Anne M. Lanthanide-binding tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography, and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1β (IL1β) and varied the length of the spacer between the LBT and the protein (denoted 1−3). Luminescence studies demonstrate that all nine constructs bind Tb3+ tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1β-S1 and IL1β-L3 constructs and for the remaining constructs by comparing the 1H−15N heteronuclear single-quantum coherence NMR spectra with that of the wild-type IL1β. Additionally, binding of LBT-loop IL1β proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loop constructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1β structure. The paramagnetic NMR spectra of loop-LBT mutant IL1β-R2 were assigned and the Δχ tensor components were calculated on the basis of RDCs and pseudocontact shifts. A structural model of the IL1β-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modeling. National Science Foundation (U.S.) (Grant MCB 0744415) Ruth L. Kirschstein National Research Service Award United States. Dept. of Energy (Offices of Biological and Environmental Research and of Basic Energy Sciences) National Institutes of Health (U.S.) (National Center for Research Resources) 2012-04-04T16:21:34Z 2012-04-04T16:21:34Z 2010-12 2010-06 Article http://purl.org/eprint/type/JournalArticle 0002-7863 1520-5126 http://hdl.handle.net/1721.1/69924 Barthelmes, Katja et al. “Engineering Encodable Lanthanide-Binding Tags into Loop Regions of Proteins.” Journal of the American Chemical Society 133.4 (2011): 808–819. https://orcid.org/0000-0002-5749-7869 en_US http://dx.doi.org/10.1021/ja104983t Journal of the American Chemical Society Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society Prof. Imperiali via Erja Kajosalo |
| spellingShingle | Barthelmes, Katja Reynolds, Anne M. Peisach, Ezra Jonker, Hendrik R. A. DeNunzio, Nicholas J. Allen, Karen N. Imperiali, Barbara Schwalbe, Harald Engineering Encodable Lanthanide-Binding Tags (LBTs) into Loop Regions of Proteins |
| title | Engineering Encodable Lanthanide-Binding Tags (LBTs) into Loop Regions of Proteins |
| title_full | Engineering Encodable Lanthanide-Binding Tags (LBTs) into Loop Regions of Proteins |
| title_fullStr | Engineering Encodable Lanthanide-Binding Tags (LBTs) into Loop Regions of Proteins |
| title_full_unstemmed | Engineering Encodable Lanthanide-Binding Tags (LBTs) into Loop Regions of Proteins |
| title_short | Engineering Encodable Lanthanide-Binding Tags (LBTs) into Loop Regions of Proteins |
| title_sort | engineering encodable lanthanide binding tags lbts into loop regions of proteins |
| url | http://hdl.handle.net/1721.1/69924 https://orcid.org/0000-0002-5749-7869 |
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