| Autor: |
Ycas PD; Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN, 55455, USA., Wagner N; Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN, 55455, USA., Olsen NM; Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN, 55455, USA., Fu R; National High Magnetic Field Lab, 1800 East Paul Dirac Dr., Tallahassee, FL, 32310, USA., Pomerantz WCK; Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN, 55455, USA. wcp@umn.edu. |
| Abstrakt: |
Incorporation of 19 F into proteins allows for the study of their molecular interactions via NMR. The study of 19 F labeled aromatic amino acids has largely focused on 4-,5-, or 6-fluorotryptophan, 4-fluorophenylalanine, (4,5, or 6FW) or 3-fluorotyrosine (3FY), whereas 2-fluorotyrosine (2FY) has remained largely understudied. Here we report a comparative analysis with different fluorinated amino acids. We first report the NMR chemical shift responsiveness of five aromatic amino acid mimics to changes in solvent polarity and find that the most responsive, a mimic of 3FY, has a 2.9-fold greater change in chemical shift compared to the other amino acid mimics in aprotic solvents including the 2FY mimic. We also probed the utility of 2FY for 19 F NMR by measuring its NMR relaxation properties in solution and the chemical shift anisotropy (CSA) of a polycrystalline sample of the amino acid by magic angle spinning. Using protein-observed fluorine NMR (PrOF NMR), we compared the influence of 2FY and 3FY incorporation on stability and pK a perturbation when incorporated into the KIX domain of CBP/p300. Lastly, we investigated the 19 F NMR response of both 2FY and 3FY-labeled proteins to a protein-protein interaction partner, MLL, and discovered that 2FY can report on allosteric interactions that are not observed with 3FY-labeling in this protein complex. The reduced perturbation to pK a and similar but reduced CSA of 2FY to 3FY supports 2FY as a suitable alternative amino acid for incorporation into large proteins for 19 F NMR analysis. |
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