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. 2024 Aug 15;128(32):7781-7791.
doi: 10.1021/acs.jpcb.4c03625. Epub 2024 Aug 6.

Translational Diffusion and Self-Association of an Intrinsically Disordered Protein κ-Casein Using NMR with Ultra-High Pulsed-Field Gradient and Time-Resolved FRET

Daria L Melnikova  1 , Venkatesh V Ranjan  2   3 , Yuri E Nesmelov  3 , Vladimir D Skirda  1 , Irina V Nesmelova  3   4
Affiliations

Translational Diffusion and Self-Association of an Intrinsically Disordered Protein κ-Casein Using NMR with Ultra-High Pulsed-Field Gradient and Time-Resolved FRET

Daria L Melnikova et al. J Phys Chem B. .

Abstract

Much attention has been given to studying the translational diffusion of globular proteins, whereas the translational diffusion of intrinsically disordered proteins (IDPs) is less studied. In this study, we investigate the translational diffusion and how it is affected by the self-association of an IDP, κ-casein, using pulsed-field gradient nuclear magnetic resonance and time-resolved Förster resonance energy transfer. Using the analysis of the shape of diffusion attenuation and the concentration dependence of κ-casein diffusion coefficients and intermolecular interactions, we demonstrate that κ-casein exhibits continuous self-association. When the volume fraction of κ-casein is below 0.08, we observe that κ-casein self-association results in a macroscopic phase separation upon storage at 4 °C. At κ-casein volume fractions above 0.08, self-association leads to the formation of labile gel-like networks without subsequent macroscopic phase separation. Unlike α-casein, which shows a strong concentration dependence and extensive gel-like network formation, only one-third of κ-casein molecules participate in the gel network at a time, resulting in a more dynamic and less extensive structure. These findings highlight the unique association properties of κ-casein, contributing to a better understanding of its behavior under various conditions and its potential role in casein micelle formation.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Diffusion attenuations of spin–echo signal in solutions of κ-casein. Diffusion attenuations, recorded at td = 50 ms, are shown for protein concentrations in the range from 0.1 to 10% (A) and 20% (B). Solutions were prepared in 100% D2O at pH 7.0. The measurements were done at 298 K. The deviation from a monoexponential attenuation is observed for all protein concentrations. Dmin decreases as the concentration of κ-casein indicated by solid blue lines drawn to 0.1 and 10% curves.
Figure 2
Figure 2
The concentration dependence of the κ-casein diffusion coefficient. The normalized concentration dependence of the normalized κ-casein diffusion coefficient ⟨D⟩ is shown by red squares. For comparison, the master curves are shown for the concentration dependence of the diffusion coefficient of linear flexible polymers (blue circles) and globular proteins (black squares). Solid lines indicate the asymptotes with the slopes of φ0 and φ–3.
Figure 3
Figure 3
The dependence of the diffusion attenuation on diffusion time in 0.5% κ-casein solution. (A) Curves 1–4 represent diffusion attenuations collected at td = 50, 150, 400, and 600 ms, respectively. (B) The dependence of the fraction of slowly diffusing κ-casein species, pmin, on td. The solid line shows the best fit of experimental data to eq 13.
Figure 4
Figure 4
The dependence of FRET efficiency on κ-casein concentration. Experimental error is shown as standard deviation from at least three independent measurements. The solid line represents the best fit of the isodesmic association model to experimental data.
Figure 5
Figure 5
The distribution of diffusion coefficients in κ-casein solutions. The diffusion coefficient spectra are shown for 0.1% (black), 1% (blue), 4% (cyan), and 10% (red).
Figure 6
Figure 6
Phase separation in κ-casein solution. (A) Image of κ-casein sample, taken at ambient room temperature immediately after removal from 4 °C, shows the separation in dilute and condensed phases. (B) The dependence of Dmax (red symbols) and Dmin (black symbols) (left vertical axis) and pmax (blue symbols, right vertical axis) on κ-casein volume fraction φ.
Figure 7
Figure 7
Diffusion attenuation for 20% κ-casein solution. (A) Curves 1–3 represent diffusion attenuations collected at td = 50, 200, and 800 ms, respectively. Curve 4 is a control. It was collected at td = 50 ms after the completion of experiments carried out at different values of td and coincides with curve 1, indicating no changes to the sample during the measurement time. (B) Curves 1–4 from panel A are replotted using coordinates log(A(g2)/A(0)) vs (γδg)2 to evaluate the dependence of the diffusion coefficient on diffusion time.
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References

    1. Anfinsen C. B. Principles that govern the folding of protein chains. Science 1973, 181, 223–230. 10.1126/science.181.4096.223. - DOI - PubMed
    1. Bellissent-Funel M. C.; Hassanali A.; Havenith M.; Henchman R.; Pohl P.; Sterpone F.; van der Spoel D.; Xu Y.; Garcia A. E. Water Determines the Structure and Dynamics of Proteins. Chem. Rev. 2016, 116, 7673–7697. 10.1021/acs.chemrev.5b00664. - DOI - PMC - PubMed
    1. Kauzmann W.Some Factors in the Interpretation of Protein Denaturation. In Advances in Protein Chemistry; Anfinsen C. B., Anson M. L., Bailey K., Edsall J. T., Eds.; Academic Press, 1959; Vol. 14, pp 1–63. - PubMed
    1. Uversky V. N.; Gillespie J. R.; Fink A. L. Why are ″natively unfolded″ proteins unstructured under physiologic conditions?. Proteins 2000, 41, 415–427. 10.1002/1097-0134(20001115)41:3<415::AID-PROT130>3.0.CO;2-7. - DOI - PubMed
    1. Mao A. H.; Lyle N.; Pappu R. V. Describing sequence-ensemble relationships for intrinsically disordered proteins. Biochem. J. 2013, 449, 307–318. 10.1042/BJ20121346. - DOI - PMC - PubMed

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