Dataset Information

Catalytic site conformations in human PNP by 19F-NMR and crystallography.

ABSTRACT: Purine nucleoside phosphorylase (PNP) is a target for leukemia, gout, and autoimmune disorders. Dynamic motion of catalytic site loops has been implicated in catalysis, but experimental evidence was lacking. We replaced catalytic site groups His257 or His64 with 6-fluoro-tryptophan (6FW) as site-specific NMR probes. Conformational adjustments in the 6FW-His257-helical and His64-6FW-loop regions were characterized in PNP phosphate-bound enzyme and in complexes with catalytic site ligands, including transition state analogs. Chemical shift and line-shape changes associated with these complexes revealed dynamic coexistence of several conformational states in these regions in phosphate-bound enzyme and altered or single conformations in other complexes. These conformations were also characterized by X-ray crystallography. Specific (19)F-Trp labels and X-ray crystallography provide multidimensional characterization of conformational states for free, catalytic, and inhibited complexes of human PNP.

SUBMITTER: Suarez J

PROVIDER: S-EPMC3584414 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Catalytic site conformations in human PNP by 19F-NMR and crystallography.

Suarez Javier J Haapalainen Antti M AM Cahill Sean M SM Ho Meng-Chiao MC Yan Funing F Almo Steven C SC Schramm Vern L VL

Chemistry & biology 20130201 2

Purine nucleoside phosphorylase (PNP) is a target for leukemia, gout, and autoimmune disorders. Dynamic motion of catalytic site loops has been implicated in catalysis, but experimental evidence was lacking. We replaced catalytic site groups His257 or His64 with 6-fluoro-tryptophan (6FW) as site-specific NMR probes. Conformational adjustments in the 6FW-His257-helical and His64-6FW-loop regions were characterized in PNP phosphate-bound enzyme and in complexes with catalytic site ligands, includi ...[more]

PMID: 23438750

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Project description:Nanometer-range distances are important for restraining the three-dimensional structure and oligomeric assembly of proteins and other biological molecules. Solid-state NMR determination of protein structures typically utilizes 13C-13C and 13C-15N distance restraints, which can only be measured up to ?7 Å because of the low gyromagnetic ratios of these nuclear spins. To extend the distance reach of NMR, one can harvest the power of 19F, whose large gyromagnetic ratio in principle allows distances up to 2 nm to be measured. However, 19F possesses large chemical shift anisotropies (CSAs) as well as large isotropic chemical shift dispersions, which pose challenges to dipolar coupling measurements. Here, we demonstrate 19F-19F distance measurements at high magnetic fields under fast magic-angle spinning (MAS) using radiofrequency-driven dipolar recoupling (RFDR). We show that 19F-19F cross-peaks for distances up to 1 nm can be readily observed in two-dimensional 19F-19F correlation spectra using less than 5 ms of RFDR mixing. This efficient 19F-19F dipolar recoupling is achieved using practically accessible MAS frequencies of 15-55 kHz, moderate 19F radio frequency field strengths, and no 1H decoupling. Experiments and simulations show that the fastest polarization transfer for aromatic fluorines with the highest distance accuracy is achieved using either fast MAS (e.g., 60 kHz) with large pulse duty cycles (>50%) or slow MAS with strong 19F pulses. Fast MAS considerably reduces relaxation losses during the RFDR ?-pulse train, making finite-pulse RFDR under fast-MAS the method of choice. Under intermediate MAS frequencies (25-40 kHz) and intermediate pulse duty cycles (15-30%), the 19F CSA tensor orientation has a quantifiable effect on the polarization transfer rate; thus, the RFDR buildup curves encode both distance and orientation information. At fast MAS, the impact of CSA orientation is minimized, allowing pure distance restraints to be extracted. We further investigate how relayed transfer and dipolar truncation in multifluorine environments affect polarization transfer. This fast-MAS 19F RFDR approach is complementary to 19F spin diffusion for distance measurements and will be the method of choice under high-field fast-MAS conditions that are increasingly important for protein structure determination by solid-state NMR.

Dataset Information

Catalytic site conformations in human PNP by 19F-NMR and crystallography.

Publications

Catalytic site conformations in human PNP by 19F-NMR and crystallography.

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