TMP-HTag

Chemical structure of the TMP-HTag linker[1]

Trimethoprim-Halotag (TMP-HTag) is a small molecule chemical linker developed for the rapid and reversible control of protein localization in living cells (Ballister). TMP is an dihydrofolate reductase (DHFR) inhibitor chosen for its specificity in binding to the bacterial form of DHFR.[1][2] The other half of the linker is a Halotag, a self labelling bacterial globular protein ligand that can bind covalently and irreversibly to the chloroalkane group of a Haloenzyme.[2][3] Positioned between the TMP group and HaloTag is a flexible linker that can be modified to optimize protein linking efficiency.[2][3] The modular structure of TMP-HaloTag makes it an ideal heterobifunctional tool for use in chemically induced dimerization (CID).[1][2] Additionally, TMP- HTag can be modified to include photo-cleavable groups that allow for the control of CID using light.[1][2]

TMP-HTag Examples

Abbr Full name
TH Trimethoprim-Halotag
CTH 7-(diethylamino)-coumarin-4-yl] methyl (DEACM)-TMP-Halo
TNH TMP-6- nitroveratryl carbamate (NVOC-HTag
TBH TMP- Benzamide-HTag
TFH TMP-Fluorobenzamide-HTag
Abbr Structural Functionality Ref
TH Linker length optimizable for efficient dimerization [1]
CTH DEACAM group on TMP end for photo cleavability that prevents off-target dimerization [3]
TNH NVOC group between TMP and HTag for photo cleavability. Requires light activation and use may result in photo toxicity. Light sensitive so must be stored and applied in controlled light. Slightly faster synthesis process than other dimerizer syntheses [3]
TBH Additional aromatic rings for rigidity that improves permeability and stability of heterodimer protein complexes that form with dimerization. Not light sensitive but slower. Lengthier synthesis but simpler  process than TNH [2]
TFH Aromatic fluorines lend lipophilicity and possibly "stickiness" that facilitates faster dimerization than with TBH. Minimizes photo-toxicity since light is not required. [2]

TMP-HTag Applications

Example Applications References
Mitotic studies [4]
Alternative lengthening of telomeres (ALT) cancer  study [5][6]
CRISPR based tool development [7]
Protein localization manipulation [2]

References

  1. ^ a b c d e Ballister, Edward R.; Aonbangkhen, Chanat; Mayo, Alyssa M.; Lampson, Michael A.; Chenoweth, David M. (2014-11-17). "Localized light-induced protein dimerization in living cells using a photocaged dimerizer". Nature Communications. 5 (1): 5475. doi:10.1038/ncomms6475. ISSN 2041-1723. PMC 4308733. PMID 25400104.
  2. ^ a b c d e f g h Lackner, Rachel M.; O'Connell, Will; Zhang, Huaiying; Chenoweth, David M. (2022-08-17). "A General Strategy for the Design and Evaluation of Heterobifunctional Tools: Applications to Protein Localization and Phase Separation". ChemBioChem. 23 (16) e202200209. doi:10.1002/cbic.202200209. ISSN 1439-4227. PMID 35599237. S2CID 248987486.
  3. ^ a b c d Zhang, Huaiying; Chenoweth, David M.; Lampson, Michael A. (2018-01-01), Maiato, Helder; Schuh, Melina (eds.), "Chapter 7 - Optogenetic control of mitosis with photocaged chemical dimerizers", Methods in Cell Biology, Mitosis and Meiosis Part A, 144, Academic Press: 157–164, doi:10.1016/bs.mcb.2018.03.006, ISBN 978-0-12-814144-1, PMID 29804668, retrieved 2023-04-03{{citation}}: CS1 maint: work parameter with ISBN (link)
  4. ^ Zhang, Huaiying; Aonbangkhen, Chanat; Tarasovetc, Ekaterina V.; Ballister, Edward R.; Chenoweth, David M.; Lampson, Michael A. (October 2017). "Optogenetic control of kinetochore function". Nature Chemical Biology. 13 (10): 1096–1101. doi:10.1038/nchembio.2456. ISSN 1552-4469. PMC 5605432. PMID 28805800.
  5. ^ Zhao, Rongwei; Chenoweth, David M.; Zhang, Huaiying (2021-04-12). "Chemical Dimerization-Induced Protein Condensates on Telomeres". JoVE (Journal of Visualized Experiments) (170) e62173. doi:10.3791/62173. ISSN 1940-087X. PMC 8118565. PMID 33900288.
  6. ^ Zhang, Huaiying; Zhao, Rongwei; Tones, Jason; Liu, Michel; Dilley, Robert L.; Chenoweth, David M.; Greenberg, Roger A.; Lampson, Michael A. (2020-08-15). Misteli, Tom (ed.). "Nuclear body phase separation drives telomere clustering in ALT cancer cells". Molecular Biology of the Cell. 31 (18): 2048–2056. doi:10.1091/mbc.E19-10-0589. ISSN 1059-1524. PMC 7543070. PMID 32579423.
  7. ^ Li, Qingyang; Gao, Yanmin; Wang, Haifeng (December 2022). "CRISPR-Based Tools for Fighting Rare Diseases". Life. 12 (12): 1968. doi:10.3390/life12121968. ISSN 2075-1729. PMC 9787644. PMID 36556333.

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