Efficient Encounter Complex Formation and Electron Transfer to Cytochrome <i>c</i> Peroxidase with an Additional, Distant Electrostatic Binding Site

Savino, Antonella Di; Foerster, Johannes M.; Haye, Thijmen La; Blok, Anneloes; Timmer, Monika; Ullmann, G. Matthias; Ubbink, Marcellus

doi:10.1002/anie.202010006

Cited by 13 publications

(28 citation statements)

References 52 publications

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“…The structures were prepared, and the Monte Carlo docking simulations were performed as described in ref . The structures of CcP and Cc from Protein Data Bank (PDB) entry 2PCC were used as a basis for the simulations.…”

Section: Methodsmentioning

confidence: 99%

“…30 CcP_B additionally has the K21E, K29E, K90E, and K97E mutations. 27 CcP_C contains the same mutations as CcP_B and D35N, E36Q, E119Q, E202Q, E210Q, E215Q, D225N, and E291Q that neutralize the charges of the WT binding site. CcP_D has mutations K75E, K76Q, N79D, Q87E, K91Q, K98Q, and N142D in comparison to CcP_A.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…The results of the kinetics measured for the Cc:CcP_A and Cc:CcP_B complexes at ionic strengths from 752 to 122 mM were presented in our previous work. 27 The same protocols were followed to measure the reactions between Cc and the other mutants of CcP. The kinetic measurements were performed at ionic strengths 752, 622, 502, 392, 292, 192, 122, and 44 mM on a SX20 stopped flow spectrometer (AppliedPhotophysics, Leatherhead, Surrey, U.K.) with a 1.2 ms dead time at room temperature following the absorbance at 416 nm, which indicates oxidation of Cc.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Kinetic Data Analysis. The stopped flow data were fitted to eq 1 as previously described: 27 i k j j j j j y…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…In a study from our groups, the charge distribution on the surface of CcP was changed strongly by addition of a negative patch on one side of CcP. 27 Monte Carlo simulations and paramagnetic relaxation enhancement (PRE) experiments showed that Cc interacts with the added patch, enlarging the surface visited in the encounter complex.…”

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confidence: 99%

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The Charge Distribution on a Protein Surface Determines Whether Productive or Futile Encounter Complexes Are Formed

et al. 2021

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Protein complex formation depends strongly on electrostatic interactions. The distribution of charges on the surface of redox proteins is often optimized by evolution to guide recognition and binding. To test the degree to which the electrostatic interactions between cytochrome c peroxidase (CcP) and cytochrome c (Cc) are optimized, we produced five CcP variants, each with a different charge distribution on the surface. Monte Carlo simulations show that the addition of negative charges attracts Cc to the new patches, and the neutralization of the charges in the regular, stereospecific binding site for Cc abolishes the electrostatic interactions in that region entirely. For CcP variants with the charges in the regular binding site intact, additional negative patches slightly enhance productive complex formation, despite disrupting the optimized charge distribution. Removal of the charges in the regular binding site results in a dramatic decrease in the complex formation rate, even in the presence of highly negative patches elsewhere on the surface. We conclude that additional charge patches can result in either productive or futile encounter complexes, depending on whether negative residues are located also in the regular binding site.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Kinetic Data Analysis. The stopped flow data were fitted to eq 1 as previously described: 27 i k j j j j j y…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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The Charge Distribution on a Protein Surface Determines Whether Productive or Futile Encounter Complexes Are Formed

et al. 2021

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Paramagnetic Chemical Probes for Studying Biological Macromolecules

et al. 2022

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Paramagnetic chemical probes have been used in electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy for more than four decades. Recent years witnessed a great increase in the variety of probes for the study of biological macromolecules (proteins, nucleic acids, and oligosaccharides). This Review aims to provide a comprehensive overview of the existing paramagnetic chemical probes, including chemical synthetic approaches, functional properties, and selected applications. Recent developments have seen, in particular, a rapid expansion of the range of lanthanoid probes with anisotropic magnetic susceptibilities for the generation of structural restraints based on residual dipolar couplings and pseudocontact shifts in solution and solid state NMR spectroscopy, mostly for protein studies. Also many new isotropic paramagnetic probes, suitable for NMR measurements of paramagnetic relaxation enhancements, as well as EPR spectroscopic studies (in particular double resonance techniques) have been developed and employed to investigate biological macromolecules. Notwithstanding the large number of reported probes, only few have found broad application and further development of probes for dedicated applications is foreseen.

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Preferential Interactions of a Crowder Protein with the Specific Binding Site of a Native Protein Complex

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et al. 2022

J. Phys. Chem. Lett.

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Nonspecific binding of crowder proteins with functional proteins is likely prevalent in vivo, yet direct quantitative evidence, let alone residue-specific information, is scarce. Here we present nuclear magnetic resonance (NMR) characterization showing that bovine serum albumin weakly but preferentially interacts with the histidine carrier protein (HPr). Notably, the binding interface overlaps with that for HPr’s specific partner protein, EIN, leading to competition. The crowder protein thus decreases the EIN-HPr binding affinity and accelerates the dissociation of the native complex. In contrast, Ficoll-70 stabilizes the native complex and slows its dissociation, as one would expect from excluded-volume and microviscosity effects. Our atomistic modeling of macromolecular crowding rationalizes the experimental data and provides quantitative insights into the energetics of protein–crowder interactions. The integrated NMR and modeling study yields benchmarks for the effects of crowded cellular environments on protein–protein specific interactions, with implications for evolution regarding how nonspecific binding can be minimized or exploited.

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Efficient Encounter Complex Formation and Electron Transfer to Cytochrome c Peroxidase with an Additional, Distant Electrostatic Binding Site

Cited by 13 publications

References 52 publications

The Charge Distribution on a Protein Surface Determines Whether Productive or Futile Encounter Complexes Are Formed

The Charge Distribution on a Protein Surface Determines Whether Productive or Futile Encounter Complexes Are Formed

Paramagnetic Chemical Probes for Studying Biological Macromolecules

Preferential Interactions of a Crowder Protein with the Specific Binding Site of a Native Protein Complex

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