Nε,Nε-Dimethyl-lysine cytochrome c as an NMR probe for lysine involvement in protein–protein complex formation

Moore, Geoffrey R.; Cox, Mark C.; Crowe, David F.; Osborne, Michael J.; Rosell, Federico I.; Bujons, Jordi; Barker, Paul D.; Mauk, Marcia R.; Mauk, A. Grant

doi:10.1042/bj3320439

Cited by 23 publications

(44 citation statements)

References 81 publications

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“…The second interaction region appears instead to be more susceptible to variations in primary sequence. In our case, the most likely additional binding site comprises helix a 5 (55)(56)(57)(58)(59)(60)(61) and the turn up to residue 64.…”

Section: Comparison With Previous Studiesmentioning

confidence: 77%

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A further investigation of the cytochrome b 5–cytochrome c complex

et al. 2003

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The interaction of reduced rabbit cytochrome b 5 with reduced yeast iso-1 cytochrome c has been studied through the analysis of 1 H-15 N HSQC spectra, of 15 N longitudinal (R 1 ) and transverse (R 2 ) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b 5 has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b 5 .

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Section: Comparison With Previous Studiesmentioning

confidence: 77%

“…1), presumably indicating that the interaction between cytochrome c and either CcP or cytochrome b 5 does not involve the same surface patch. NMR data on a derivative of horse heart cytochrome c where Lys residues had been dimethylated also support this hypothesis [61].…”

Section: Comparison With Previous Studiesmentioning

confidence: 86%

A further investigation of the cytochrome b 5–cytochrome c complex

et al. 2003

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“…The presence of multiple orientations within Cc-CcP complex previously has been suggested by several studies (9)(10)(11)(12)(13)(14)(15)17). A complex comprising an equilibrium between a well defined form and a dynamic state (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For the control experiment with low Cc concentration, described in Results and Discussion, samples of 0.3 mM CcP-MTS or CcP-MTSL with 0.06 mM 15 N Cc were used. The pH of the samples was adjusted to 6.00 Ϯ 0.05 with small aliquots of 0.1 M HCl or 0.1 M NaOH.…”

Section: Materials and Methods Protein Preparation Isotopically Enrimentioning

confidence: 99%

“…However, it has remained a matter of debate whether this structure represents the only form in solution (6)(7)(8). According to several studies, the complex is dynamic, and the crystal structure might represent only a subpopulation of protein orientations (9)(10)(11)(12)(13)(14)(15). Recent studies show that the photoinduced ET between Znsubstituted CcP and Cc, both in the crystal (8,16) and in solution (17), occurs with faster backward than forward rates, indicating that the complex is present in multiple forms, only a few of which are ET-active (17).…”

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confidence: 99%

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Solution structure and dynamics of the complex between cytochromecand cytochromecperoxidase determined by paramagnetic NMR

Volkov

Worrall

Holtzmann

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

244

361

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The physiological complex of yeast cytochrome c peroxidase and iso-1-cytochrome c is a paradigm for biological electron transfer. Using paramagnetic NMR spectroscopy, we have determined the conformation of the protein complex in solution, which is shown to be very similar to that observed in the crystal structure [ electron transfer ͉ encounter state ͉ transient complex ͉ spin label ͉ paramagnetic relaxation enhancement T he process of protein complex formation can be described by a two-step model, in which a short-lived, dynamic encounter complex precedes a dominant, well defined state (Fig. 1). The former enables proteins to undergo reduced-dimensionality search of the optimal binding geometry, thereby accelerating molecular association as compared with 3D diffusion (1). Fast molecular association is essential for protein-protein complexes that require high turnover rates, like those involved in electron transfer (ET) in photosynthesis, respiration, and other metabolic processes (2). The physiological complex of yeast iso-1-cytochrome c (Cc) and yeast cytochrome c peroxidase (CcP) is a paradigm for the intermolecular ET (3) and is one of the few transient ET complexes for which a crystal structure has been solved (4). A recent study has confirmed that the protein-protein orientation observed in the crystal is ET-active (5). However, it has remained a matter of debate whether this structure represents the only form in solution (6-8). According to several studies, the complex is dynamic, and the crystal structure might represent only a subpopulation of protein orientations (9-15). Recent studies show that the photoinduced ET between Znsubstituted CcP and Cc, both in the crystal (8, 16) and in solution (17), occurs with faster backward than forward rates, indicating that the complex is present in multiple forms, only a few of which are .Characterization of the binding interface in the dynamic encounter state (Fig. 1B) has so far proven to be elusive. Investigation of protein complexes by using x-ray crystallography or conventional NMR spectroscopy addresses only the singleorientation species (Fig. 1C), and the only way to visualize the dynamic state is offered by theoretical modeling studies (11,18). In the recent, elegant work of Clore and coworkers (19,20), it was shown how paramagnetic relaxation can be applied to study the dynamic state of protein-DNA complexes. We report on the application of an analogous experimental approach that allows us to define both the dominant protein-protein orientation (Fig. 1C) and the conformational space sampled by the proteins in the dynamic encounter complex (Fig. 1B). Results and DiscussionSolution Structure of the Complex. The concept of the approach is that NMR resonance intensities of one of the proteins in the complex are affected by a paramagnetic spin label covalently attached to the other protein (Fig. 2). The paramagnetic effects are converted into distance restraints (21-23), which can be used to calculate protein-protein orientations within the complex (24). Five ...

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Protein–protein docking of electron transfer complexes: Cytochromecoxidase and cytochromec

Flöck

Helms

2002

Proteins

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Electron transferring protein complexes form only transiently and the crystal structures of electron transfer protein--protein complexes involving cytochrome c could so far be determined only for the pairs of yeast cytochrome c peroxidase (CcP) with iso-1-cytochrome c (iso-1-cyt c) and with horse heart cytochrome c (cyt c). This article presents models from computational docking for complexes of cytochrome c oxidase (COX) from Paracoccus denitrificans with horse heart cytochrome c, and with its physiological counterpart cytochrome c552 (c552). Initial docking is performed with the FTDOCK program, which permits an exhaustive search of translational and rotational space. A filtering procedure is then applied to reduce the number of complexes to a manageable number. In a final step of structural and energetic refinement, the complexes are optimized by rigid-body energy minimization with the molecular mechanics package CHARMM. This methodology was first tested on the CcP:iso-1-cyt c complex, in which the complex with the lowest CHARMM energy has an RMSD from the crystal structure of only 1.8 A (C(alpha) carbon atoms). Notably, the crystal conformation has an even lower energy. The same procedure was then applied to COX:cyt c and COX:c552. The lowest-energy COX:cyt c complex is very similar to a docking model previously described for the complex of bovine cytochrome c oxidase with horse heart cytochrome c. For the COX:c552 complex, cytochrome c552 is found in two different orientations, depending on whether it is docked against COX from a two-subunit or from a four-subunit crystal structure, respectively. Both conformations are discussed critically in the light of the available experimental data.

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Nε,Nε-Dimethyl-lysine cytochrome c as an NMR probe for lysine involvement in protein–protein complex formation

Cited by 23 publications

References 81 publications

A further investigation of the cytochrome b 5–cytochrome c complex

A further investigation of the cytochrome b 5–cytochrome c complex

Solution structure and dynamics of the complex between cytochromecand cytochromecperoxidase determined by paramagnetic NMR

Protein–protein docking of electron transfer complexes: Cytochromecoxidase and cytochromec

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