Abstract We developed a new method to elucidate the binding kinetics kon and koff, and the dissociation constant KD (=koff/kon), of protein-protein interactions without observable bound resonances of the protein of interest due to high molecular weight in a complex with a large target protein. In our method, kon and koff rates are calculated from the analysis of longitudinal relaxation rates of free resonances measured for multiple samples containing different concentration ratios of 15N-labeled protein and substoichiometric amounts of the target protein. The method is applicable to interactions that cannot be analyzed by relaxation dispersion spectroscopy due to slow interactions on millisecond to second timescale and/or minimal conformational (chemical shift) change upon binding. We applied the method to binding of the B1 domain of protein G (GB1) to immunoglobulin G, and derived the binding kinetics despite the absence of observable bound GB1 resonances.
Content Type Journal Article
Pages 1-9
DOI 10.1007/s10858-011-9511-7
Authors
Kenji Sugase, Bioorganic Research Institute, Suntory Foundation for Life Sciences, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka, 618-8503 Japan
Elucidating slow binding kinetics of a protein without observable bound resonances by longitudinal relaxation NMR spectroscopy.
Elucidating slow binding kinetics of a protein without observable bound resonances by longitudinal relaxation NMR spectroscopy.
Elucidating slow binding kinetics of a protein without observable bound resonances by longitudinal relaxation NMR spectroscopy.
J Biomol NMR. 2011 May 28;
Authors: Sugase K
We developed a new method to elucidate the binding kinetics k(on) and k(off), and the dissociation constant K(D) (=k(off)/k(on)), of protein-protein interactions without observable bound resonances of the protein of interest due to high molecular...
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06-01-2011 02:30 PM
Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion [Biophysics and Computational Biology]
Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion
Meinhold, D. W., Wright, P. E....
Date: 2011-05-31
Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use 15N, , and 13CO NMR R2 relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which...
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Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion.
Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion.
Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion.
Proc Natl Acad Sci U S A. 2011 May 11;
Authors: Meinhold DW, Wright PE
Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use (15)N, , and (13)CO NMR R(2)...
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05-13-2011 02:40 PM
Structure, Dynamics, and Kinetics of Weak Protein-Protein Complexes from NMR Spin Relaxation Measurements of Titrated Solutions.
Structure, Dynamics, and Kinetics of Weak Protein-Protein Complexes from NMR Spin Relaxation Measurements of Titrated Solutions.
Structure, Dynamics, and Kinetics of Weak Protein-Protein Complexes from NMR Spin Relaxation Measurements of Titrated Solutions.
Angew Chem Int Ed Engl. 2011 Mar 18;
Authors: Salmon L, Ortega Roldan JL, Lescop E, Licinio A, van Nuland N, Jensen MR, Blackledge M
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03-23-2011 05:41 PM
[NMR paper] NMR spectroscopic detection of protein protons and longitudinal relaxation rates betw
NMR spectroscopic detection of protein protons and longitudinal relaxation rates between 0.01 and 50 MHz.
Related Articles NMR spectroscopic detection of protein protons and longitudinal relaxation rates between 0.01 and 50 MHz.
Angew Chem Int Ed Engl. 2005 Apr 8;44(15):2223-5
Authors: Bertini I, Gupta YK, Luchinat C, Parigi G, Schlörb C, Schwalbe H
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[NMR paper] Longitudinal (1)H relaxation optimization in TROSY NMR spectroscopy.
Longitudinal (1)H relaxation optimization in TROSY NMR spectroscopy.
Related Articles Longitudinal (1)H relaxation optimization in TROSY NMR spectroscopy.
J Am Chem Soc. 2002 Oct 30;124(43):12898-902
Authors: Pervushin K, Vögeli B, Eletsky A
A general method to enhance the sensitivity of the multidimensional NMR experiments performed at high-polarizing magnetic field via the significant reduction of the longitudinal proton relaxation times is described. The method is based on the use of two vast pools of "thermal bath" 1H spins residing on...
Elucidating slow binding kinetics of a protein without observable bound resonances by longitudinal relaxation NMR spectroscopy
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