Fluorotyrosine alkaline phosphatase: internal mobility of individual tyrosines and the role of chemical shift anisotropy as a 19F nuclear spin relaxation mechanism in proteins.

19 F nuclear spin relaxation data for fluorotyrosine alkaline phosphatase at 94 and 235 MHz are presented. The factor of 2·5 in increased chemical shift separation expected at the higher field was almost exactly matched by an increase in 19 F linewidths due to the field-dependent relaxation mechanism of chemical shift anisotropy. Consequently, no significant improvement in resolution or signal/noise was gained at the higher field. At 235 MHz the chemical shift anisotropy mechanism produces about 50% of the observed linewidths but only 10 to 20% at 94 MHz. Using a density matrix formalism, complete expressions are derived for the relaxation of a nuclear spin with an asymmetric chemical shift tensor (three independent principal elements) undergoing axially symmetric (symmetric top) diffusion. With this formalism, the chemical shift anisotropy contributions to T 1 and T 2 for fluorine-labeled residues undergoing one link of internal motion may be calculated. Such a calculation requires detailed knowledge of both the magnitude and orientation within the molecular frame of the three principal elements of the traceless chemical shift tensor. Data from the literature on 19 F chemical shift tensors are collected and used in calculations of chemical shift anisotropy relaxation for m -fluorotyrosine, p -fluorophenylalanine, and trifluoromethyl residues incorporated in proteins. The relaxation is sensitive to internal motion about the C β −C γ bond in the aromatic residues and the C 3 axis of the CF 3 group. It is found that the contribution of chemical shift anisotropy to T 1 will be negligible except when dipolar relaxation is particularly inefficient and internal motion is near the Larmor frequence ω 0 . The contribution of chemical shift anisotropy to the linewidth increases with ω 0 2 and becomes significant for proteins with M r >20,000 and fields >50 kgauss. For fluorotyrosine alkaline phosphatase the calculated chemical shift anisotropy linewidths as a function of internal motion about C β −C γ span a range of values that encompasses the observed linewidths at 235 MHz for nine of the eleven fluorotyrosine residues. Two other fluorotyrosines exhibit linewidths with an even greater field dependence, suggesting the influence of chemical exchange ( via slow internal rotation) between different magnetic environments. Combining dipolar T 1 and linewidth data, chemical shift anisotropy linewidth data, and the nuclear Overhauser enhancement observed when irradiating protein protons, it is possible to derive the overall tumbling correlation time τ c for alkaline phosphatase and place limits on the internal mobility of the tyrosine residues. Thus, for overall tumbling we find that τ c =76·0±15 ns or D macro =2·2×10 6 s −1 . For rotation about C α −C β , D int ( α−β )≤10 6 s −1 for all tyrosines. Rotation about C β −C γ has D int ( β−γ )≤10 8 s −1 for all tyrosines, with nine tyrosines probably in the range 10 6 to 10 8 s −1 and two others in the range 10 2 to 10 5 s −1 . These results are compared with other literature data concerning protein mobility.