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Tryptophan plays a critical role in proteins by contributing to stability, allostery, and catalysis. Using fluorine ( 19 F) nuclear magnetic resonance (NMR), protein conformational dynamics and structure-activity relationships (SARs) can be studied via fluorotryptophan reporters. Tryptophan analogs such as 4-, 5-, 6-, or 7-fluorotryptophan can be routinely incorporated into proteins during heterologous expression by arresting endogenous tryptophan biosynthesis. Building upon the large 19 F chemical shift dispersion associated with 5-fluorotryptophan, we introduce an approach to the incorporation of 13 C-enriched 5-fluorotryptophan using a direct biosynthetic precursor, 5-fluoroanthranilic acid-(phenyl- 13 C 6 ). The homodimeric enzyme fluoroacetate dehalogenase (FAcD), a thermophilic alpha/beta hydrolase responsible for the hydrolysis of a C-F bond in fluoroacetate, was expressed and biosynthetically labeled with (phenyl- 13 C 6 ) 5-fluorotryptophan. The resulting two-dimensional 19 F- 13 C (transverse relaxation optimized spectroscopy) TROSY heteronuclear correlation spectra provide complete resolution of all 9 tryptophan residues in the apo enzyme and FAcD saturated with the substrate analog bromoacetate. The ( 19 F, 13 C) correlation spectra also reveal a multitude of minor resonances in the apo sample. The role of each tryptophan residue in allosteric communication was validated with computational rigidity transmission allostery analysis, which in this case explores the relative interprotomer communication between all possible tryptophan pairs., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: To characterize anterior corneal higher-order aberration (HOA) excimer ablation map patterns in postoperative LASIK ectasia (POE) and to examine correlations between newly identified corneal HOA ablation map features of POE and known topographic indices., Methods: Prospective multicenter non-interventional descriptive study. A total of 28 eyes from 22 POE patients were enrolled. The postoperative HOA ablation map was derived from Topolyzer Vario corneal imaging at the time of POE diagnosis. Features that recurred were identified and then analyzed. Correlations to Orbscan indices were studied., Results: An arrangement of two elliptical paracentral ablation islands, deep inferior and shallow superior, in direct mirror-like opposition to each other, were identified on all HOA maps. The paracentral islands were accompanied by peripheral ablation crescents. The deep paracentral inferior island 'hot spot' coincided with the topographical apical POE cone and was highly reproducible in angular position (249.3 ± 17.3°). There was significant variation in ablation depth (shallow superior island: 11.5 ± 6.9 μm and deep inferior island: 32.5 ± 18.8 μm). The superior crescents had high variability in depth (34.8 ± 18.9 μm). Strong correlations were found between the corneal irregularity index and the ablation depth difference between the deep and shallow paracentral islands (R = 0.96; P < 0.0001)., Conclusion: The corneal HOA excimer ablation map revealed a recurring, distinct, easily recognizable pattern in POE eyes. Validated Orbscan POE indices and HOA ablation map islands showed a strong correlation. It is possible to extract useful information from the corneal HOA ablation map, potentially making it suitable for diagnosing and monitoring POE although more studies are needed., (© 2023. The Author(s).)