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, 112 (8), 1586-1596

Effect of Phosphorylation on a Human-like Osteopontin Peptide

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Effect of Phosphorylation on a Human-like Osteopontin Peptide

Samuel Lenton et al. Biophys J.

Abstract

The last decade established that the dynamic properties of the phosphoproteome are central to function and its modulation. The temporal dimension of phosphorylation effects remains nonetheless poorly understood, particularly for intrinsically disordered proteins. Osteopontin, selected for this study due to its key role in biomineralization, is expressed in many species and tissues to play a range of distinct roles. A notable property of highly phosphorylated isoforms of osteopontin is their ability to sequester nanoclusters of calcium phosphate to form a core-shell structure, in a fluid that is supersaturated but stable. In Biology, this process enables soft and hard tissues to coexist in the same organism with relative ease. Here, we extend our understanding of the effect of phosphorylation on a disordered protein, the recombinant human-like osteopontin rOPN. The solution structures of the phosphorylated and unphosphorylated rOPN were investigated by small-angle x-ray scattering and no significant changes were detected on the radius of gyration or maximum interatomic distance. The picosecond-to-nanosecond dynamics of the hydrated powders of the two rOPN forms were further compared by elastic and quasi-elastic incoherent neutron scattering. Phosphorylation was found to block some nanosecond side-chain motions while increasing the flexibility of other side chains on the faster timescale. Phosphorylation can thus selectively change the dynamic behavior of even a highly disordered protein such as osteopontin. Through such an effect on rOPN, phosphorylation can direct allosteric mechanisms, interactions with substrates, cofactors and, in this case, amorphous or crystalline biominerals.

Figures

Figure 1
Figure 1
Guinier plots for phosphorylated (A) and unphosphorylated (B) rOPN. Rg and I0 were recovered from the fits of the straight lines where (q × Rg) ≤ 1.3. (C) Expected Rg values for chemically denatured proteins were determined using Eq. 3, where the region between dashed lines represents the confidence interval of the Flory equation. The diamonds show experimental Rg obtained for phosphorylated (darker) and unphosphorylated rOPN (lighter). The lower continuous gray line shows the expected Rg value for globular proteins. (D) P(r) distributions of phosphorylated (black) and unphosphorylated (gray) rOPN, from which the Rg and Dmax were obtained. (E) Kratky plots are shown (same color scheme as D).
Figure 2
Figure 2
Ensemble optimization analysis of the HPLC-SAXS profile measured for rOPN peptides. (A) Rg and (B) Dmax distribution for the random ensemble (solid black line), phosphorylated rOPN (dashed), and unphosphorylated rOPN (dots) is shown. The fits to the corresponding scattering curves are shown in (C) for phosphorylated rOPN and (D) unphosphorylated rOPN.
Figure 3
Figure 3
Apparent MSDs of the two forms of rOPN, obtained from polynomial non-Gaussian fits to elastic fixed window temperature scans performed: (A) on the IRIS and (B) on the IN16B spectrometers. (Both insets) Elastic scattering intensities as a function of q2, at 305 K, are given. The point symbols show data binned along the temperature-axis to 5 K-intervals. The error bars correspond to 1 SD of uncertainty. Note that on the (B) inset, the error bars are smaller than the point symbols.
Figure 4
Figure 4
Example QENS spectra measured on IN16B (q = 1.7 Å−1), and q-wise fit (Eq. 4) at two temperatures, for unphosphorylated (uP) and phosphorylated (P) rOPN, normalized to their maximum for a better comparison. For clarity, the data are rebinned for |ħω| > 1 μeV. The wings of the spectra—|ħω| > 15 μeV—are the same for both rOPN forms at both temperatures, within the error bars (1 SD of uncertainty).
Figure 5
Figure 5
Comparison of the widths for the two rOPN forms. (A) From fits to IN16B spectra: for 200 and 250 K data, the fits (Eq. 4) are straight lines. For the 280 K data, a jump diffusion model was used (Eq. 5). The points at the two highest q values are not shown (signal-to-noise ratio too high to be included in a reliable data analysis). (B) IRIS spectra and corresponding fits (Eq. 4) at 300 K are given. The lines are guides to the eye. The error bars correspond to 1 SD of uncertainty.
Figure 6
Figure 6
EISF, A0, for the two forms of rOPN from fits (lines) by Eq. 7. The fraction of immobile atoms, at the resolution times of each spectrometer, is shown for each plateau: (A) IN16B QENS spectra (∼4 ns); see also Fig. S11 for more information on the fit parameters. (B) IRIS spectra (∼15 ps). The effective radius a is 2.63 ± 0.86 Å and 2.71 ± 0.36 Å for unphosphorylated and phosphorylated rOPN, respectively. The data point error bars correspond to 1 SD of uncertainty.

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