Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redoxp roteins is often optimizedt os teer ar edox partner to the electron transfer active binding site.T ot est whether the optimizeddistribution is more important than the strength of the electrostatic interactions,anadditional negative patch was introduced on the surface of cytochrome cp eroxidase,a way from the stereospecific binding site,a nd its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner,c ytochrome c, interacts with the new patch.U nexpectedly,t he rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimizedc harge distribution.

Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redoxp roteins is often optimizedt os teer ar edox partner to the electron transfer active binding site.T ot est whether the optimizeddistribution is more important than the strength of the electrostatic interactions,anadditional negative patch was introduced on the surface of cytochrome cp eroxidase,a way from the stereospecific binding site,a nd its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner,c ytochrome c, interacts with the new patch.U nexpectedly,t he rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimizedc harge distribution.

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.

Paramagnetic NMR methods are excellently suited for the study of protein-protein complexes in solution. Intermolecular pseudocontact shifts (PCSs), residual dipolar couplings (RDCs) and paramagnetic relaxations enhancements (PRE) can be used, ideally in combination, for docking proteins and determining their orientation in the complex. PCSs can be used for breaking the structure symmetry in dimer complexes. PCSs also can be applied to detect structural differences in proteins and protein complexes in solution in comparison to crystal structures. RDCs are sensitive to the degree of alignment of both partners in a protein complex and are thus very useful to detect dynamics within complexes. PREs can detect states in which nuclei approach a paramagnetic centre closely, even if it exists only for a small fraction of the time. Thus, PREs are used to detect minor states and characterize ensembles.PRE studies have been the foundation for characterizing encounter states and the process of protein complex formation. In weak complexes, such as found in electron transfer chains, proteins can be in an encounter state for a large fraction of the complex lifetime.Paramagnetic NMR tools thus have found many applications for studying protein complexes, and more may be on the horizon.

We present a novel approach to study transient protein-protein complexes with standard, 9 GHz, and high-field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso-1-cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfo-nate] attached at position 81 of Cc (SLÀ Cc). A dissociation constant K D of 20 � 4 × 10 À 6 M (EPR and NMR) and an equal amount of stereo-specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc-interaction surface, with the stereo-specific complex.