NMR Studies of Paramagnetic Systems to Characterise Small Molecule:Protein and Protein:Protein Interactions

Moore, Geoffrey R.; Cox, Mark C.; Crowe, David F.; Osborne, Michael J.; Mauk, A. Grant; Wilson, Moira

doi:10.1007/978-94-015-8573-6_5

Cited by 3 publications

(1 citation statement)

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“…To learn more about the stoichiometry of the protein complex and possible location of the two binding sites, we used 1 H NMR spectroscopy. Although the interactions between the binary complex and an additional molecule of cytochrome c are only transient, they can be detected in 1 H NMR titration experiments …”

Section: Discussionmentioning

confidence: 99%

Protein Docking and Gated Electron-Transfer Reactions between Zinc Cytochrome c and the New Plastocyanin from the Fern Dryopteris crassirhizoma. Direct Kinetic Evidence for Multiple Binary Complexes

et al. 2000

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A new plastocyanin from the fern Dryopteris crassirhizoma markedly differs from other plastocyanins in having a very large acidic surface, which extends into the area that is hydrophobic in other plastocyanins. The exceptionally large dipole moment of 439 D has a completely different orientation and protrudes through the “northwest” region of the surface, which is now acidic. Consequently, the new plastocyanin differs from its congeners in the photoinduced reaction with zinc cytochrome c: 3Zncyt + pc(II) → Zncyt+ + pc(I). At ionic strength ≤20 mM and solution viscosity ≤1.8 cp, at least three exponentials are needed to describe the oxidative quenching of 3Zncyt. Besides a bimolecular phase, there are two distinct unimolecular phases corresponding to electron transfer within two different persistent complexes 3Zncyt/pc(II). So-called normal and reverse titrations yield consistent values of the unimolecular rate constants: k 1 is (3.3 ± 0.7) × 105 s-1 and (3.2 ± 0.4) × 105 s-1, and k 2 is (7.6 ± 0.8) × 103 s-1 and (8.2 ± 1.2) × 103 s-1. The respective ΔH ⧧ values also differ (16 ± 2 and 27 ± 7 kJ/mol), but ΔS ⧧ values are the same (−88 ± 7 and −78 ± 23 J/K mol). Viscosity effects and also unrealistic reorganizational energies obtained in fittings of temperature effects to Marcus theory reveal that both unimolecular electron-transfer reactions (k 1 and k 2) are gated by structural rearrangement of the respective binary complexes. Additional evidence for multiple persistent binary complexes is dependence on ionic strength of the apparent rate constant k app for electron transfer in the transient binary complex 3Zncyt/pc(II). Analysis of this dependence indicates that rearrangement of the protein complexes involves relatively large migration of zinc cytochrome c, which is facilitated at higher ionic strength. When zinc cytochrome c is present in excess, a transient, but not persistent, ternary complex Zncyt/pc/Zncyt is formed; both reverse titration and analysis of the effects of protein association on the 1H NMR chemical shifts support this conclusion. Existence of a ternary complex is consistent with the existence of multiple binary complexes. Monte Carlo simulations show possible docking configurations of the binary Zncyt/pc complexes. These theoretical calculations, in conjunction with our kinetic data, suggest that the faster (k 1) and slower (k 2) intracomplex reactions seem to occur when 3Zncyt docks, respectively, in the “northeast” and “northwest” surface regions of fern plastocyanin (in the conventional orientation). The new type of docking, on the “northwest” side of the plastocyanin surface, is favored by new acidic residues in this region.

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Section: Discussionmentioning

confidence: 99%