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. 2020:2141:663-681.
doi: 10.1007/978-1-0716-0524-0_34.

Determining Binding Kinetics of Intrinsically Disordered Proteins by NMR Spectroscopy

Ke Yang  1 , Munehito Arai  2 , Peter E Wright  3
Affiliations

Determining Binding Kinetics of Intrinsically Disordered Proteins by NMR Spectroscopy

Ke Yang et al. Methods Mol Biol. 2020.

Abstract

The unique structural flexibility of intrinsically disordered proteins (IDPs) is central to their diverse functions in cellular processes. Protein-protein interactions involving IDPs are frequently transient and dynamic in nature. Nuclear magnetic resonance (NMR) spectroscopy is an especially powerful tool for characterizing the structural propensities, dynamics, and interactions of IDPs. Here we describe applications of the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiment in combination with NMR titrations to characterize the kinetics and mechanisms of interactions between intrinsically disordered proteins and their targets. We illustrate the method with reference to interactions between the activation domain of the human T-cell leukemia virus type-I (HTLV-1) basic leucine zipper protein (HBZ) and its cellular binding partner, the KIX domain of the transcriptional coactivator CBP.

Keywords: CPMG; IDP; Protein dynamics; Protein interaction; Relaxation dispersion.

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Figures

Fig. 1.
Fig. 1.. CPMG relaxation dispersion experiment.
Simulation showing a peak that broadens at lower refocusing pulse frequencies (longer τCP delays) and the effective transverse relaxation rates (R2eff) as a function of pulsing frequency. R20 represents the intrinsic transverse relaxation rate that can be measured at high pulsing frequencies. Rex can be calculated by extracting R20 from experimentally determined R2eff.
Fig. 2.
Fig. 2.. Representative 15N relaxation dispersion profiles of HBZ at [KIX]:[HBZ] = 0.02:1.
Dispersion data for 15N-labeled HBZ were collected at 15N frequencies of 50.65 MHz (black dots, collected on a 500 MHz spectrometer) and 81.20 MHz (red dots, collected on an 800 MHz spectrometer). The lines represent the fits of the relaxation dispersion profiles for each individual residue.
Fig. 3.
Fig. 3.
Overview of workflow of dispersion data fitting using GLOVE.
Fig. 4.
Fig. 4.. Globally fitted 15N relaxation dispersion titration profiles of HBZ.
Superposition of dispersion curves for 15N-labeled HBZ for all titration points, with [KIX]:[HBZ] ratios of 0.010:1, 0.015:1, 0.020:1, and 0.025:1. Dispersion data were acquired on 500-MHz (15N frequencies of 50.65 MHz) and 800-MHz (15N frequencies of 81.20 MHz) spectrometers.
Fig. 5.
Fig. 5.. Schematic representation of three-state binding models.
(a) Three-state binding model where there are two free forms and one bound state. L and L* (colored in blue) represent two exchanging forms of the free ligand. The protein-ligand complex is shown as [P:L] (colored in pink). k12 and k21 are rate constants of the forward and reverse reaction between the two free forms of ligand. ΔωF1F2 represents the chemical shift difference between the two free forms. ΔωF1B is the chemical shift difference between the bound and the most highly populated form of the free ligand (L). (b) Three-state binding model where there is one free form (L, blue) and two bound forms (P:L* and P:L, pink). k12 and k21 are rate constants for the exchange between the two bound forms. ΔωFB1 and ΔωFB2 are the chemical shift differences between the free form and the two bound forms.
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References

    1. Wright PE, Dyson HJ (2015) Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 16 (1):18–29. doi:10.1038/nrm3920 - DOI - PMC - PubMed
    1. Schneider R, Maurin D, Communie G et al. (2015) Visualizing the molecular recognition trajectory of an intrinsically disordered protein using multinuclear relaxation dispersion NMR. J Am Chem Soc 137 (3):1220–1229. doi:10.1021/ja511066q - DOI - PubMed
    1. Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447 (7147):1021–1025. doi:10.1038/nature05858 - DOI - PubMed
    1. Loria JP, Rance M, Palmer AG (1999) A relaxation-compensated Carr-Purcell-Meiboom-Gill sequence for characterizing chemical exchange by NMR spectroscopy. J Am Chem Soc 121 (10):2331–2332. doi:DOI 10.1021/ja983961a - DOI
    1. Trott O, Palmer AG 3rd, (2002) R1rho relaxation outside of the fast-exchange limit. J Magn Reson 154 (1):157–160. doi:10.1006/jmre.2001.2466 - DOI - PubMed

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