Assessing the applicability of 19F labeled tryptophan residues to quantify protein dynamics

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to study the dynamics of biomolecules in solution. Most NMR studies exploit the spins of proton, carbon and nitrogen isotopes, as these atoms are highly abundant in proteins and nucleic acids. As an alternative and complementary approach, fluorine atoms can be introduced into biomolecules at specific sites of interest. These labels can then be used as sensitive probes for biomolecular structure, dynamics or interactions. Here, we address if the replacement of tryptophan with 5-fluorotryptophan residues has an effect on the overall dynamics of proteins and if the introduced fluorine probe is able to accurately report on global exchange processes. For the four different model proteins (KIX, Dcp1, Dcp2 and DcpS) that we examined, we established that 15N CPMG relaxation dispersion or EXSY profiles are not affected by the 5-fluorotryptophan, indicating that this replacement of a proton with a fluorine has no effect on the protein motions. However, we found that the motions that the 5-fluorotryptophan reports on can be significantly faster than the backbone motions. This implies that care needs to be taken when interpreting fluorine relaxation data in terms of global protein motions. In summary, our results underscore the great potential of fluorine NMR methods, but also highlight potential pitfalls that need to be considered.

4D structural biology: quantitative dynamics in the eukaryotic RNA exosome complex

“…The methyl-TROSY methods that we deployed are "blind" to regions that are devoid of Ile or Met residues. To also investigate such regions, we turned to 19 F NMR methods that were recently shown to be excellent tools to study interactions and dynamics on a broad range of timescales (40)(41)(42)(43), even for larger complexes (44)(45)(46)(47). First, we employed amber codon suppression to introduce a 4trifluoromethyl-L-phenylalanine (tfmF) into Rrp41 at position D113 (Rrp41 D113tfmF ).…”

Identifying Exchangeable Protons in a 1D NMR Spectrum by Spatially Selective Exchange‐Editing

“…[6][7][8][9] Exchange NMR spectroscopy and its variants are frequently used techniques and can be applied to a diverse set of problems including e. g. lithium ion dynamics in electrode materials, investigation of organic reaction mechanisms and studying protein dynamics. [10][11][12] All these methods take some time, either because of additional sample handling or the acquisition of two-dimensional datasets. The time problem could be addressed by 2D ultrafast NMR (UF-NMR) which can enable the recording of a full 2D experiment in a single scan.…”

“…Related approaches which have recently attracted interest are relaxation exchange NMR spectroscopy (REXSY) and diffusion exchange NMR spectroscopy (DEXSY) which use difference in T 2 – relaxation rate and difference in diffusion coefficient as contrast respectively [6–9] . Exchange NMR spectroscopy and its variants are frequently used techniques and can be applied to a diverse set of problems including e. g. lithium ion dynamics in electrode materials, investigation of organic reaction mechanisms and studying protein dynamics [10–12] …”

Decorating phenylalanine side-chains with triple labeled 13 C/ 19 F/ 2 H isotope patterns

“…Despite of the great potential of the 19 F- 13 C TROSY technique to characterize structural dynamics and interactions of large biomolecules, corresponding methods for e ciently introducing 19 F- 13 C spin systems in the target biomolecules are still poorly developed. Notable exceptions contain the application of [3,[5][6][7][8][9][10][11][12][13] C 2 ]-3uorotyrosine (Fig. 1A; Boeszoermenyi et al 2019), various 13 C-uoroindoles (e.g.…”