Identification of productive and futile encounters in an electron transfer protein complex

Abstract

Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble of more dynamic orientations: the encounter states. The structure of the stereospecific complex between cytochrome P450cam and putidaredoxin was solved recently by X-ray diffraction as well as paramagnetic NMR spectroscopy. Other than the stereospecific complex, the NMR data clearly show the presence of additional states in the complex in solution. In these encounter states, populated for a small percentage of the time, putidaredoxin assumes multiple orientations and samples a large part of the surface of cytochrome P450cam. To characterize the nature of the encounter states, an extensive paramagnetic NMR dataset has been analyzed using the Maximum Occurrence of Regions methodology. The analysis reveals the location and maximal spatial extent of the additional states needed to fully explain the NMR data. Under the assumption of sparsity of the size of the conformational ensemble, several minor states can be located quite precisely. The distribution of these minor states correlates with the electrostatic potential map around cytochrome P450cam. Whereas some minor states are on isolated positively charged patches, others are connected to the stereospecific site via positively charged paths. The existence of electrostatically favorable pathways between the stereospecific interaction site and the different minor states or lack thereof suggests a means to discriminate between productive and futile encounter states.paramagnetic NMR | encounter complex | cytochrome P450cam | putidaredoxin | maximum occurrence C rystal structures suggest that proteins assume unique, stereospecific orientations within protein-protein complexes. However, a number of studies in solution have made clear that encounter states are an inherent element of protein complexes (1-8), especially in electron transfer (ET), where the interactions are often extremely fast (9). In the encounter complex, the proteins assume multiple other orientations, often in equilibrium with the major stereospecific state. In low-affinity complexes with dissociation constant (K d ) values > 10 μM, the encounter complex can represent a sizeable fraction, and in some cases, a well-defined, stereospecific complex may even be absent (10-15). The presence of encounter states may be a consequence of the chemical nature of proteins. In nonobligate stereospecific complexes, the interface represents a small fraction of the total protein surface, and therefore, it is reasonable to assume that weak interactions also occur elsewhere. In the case in which protein pairs have evolved to exhibit a high association rate by using electrostatic preorientation, electrostatic patches seem to enhance the presence of encounter states (16,17). On the one hand, the preorientation reduces the surface area that is visited by the partner, thus enhancing the number of productive encounters, but on the other hand, highly charged patches can bind the oppositely charged protein in many orientations with a...

Controlling Redox Enzyme Orientation at Planar Electrodes

“…This electrostatic interaction allows both partners to come to a close distance which then induces fast intermolecular ET. Various other striking examples of electrostatic-driven protein-protein interactions for efficient ET are documented in the literature: interaction between cytochrome c oxidase and cytochrome c [45], sulfite reductase and ferredoxin [46], cytochrome P450 and putidaredoxin [47], and NADH-cytochrome b 5 reductase and Fe(III)-cytochrome b 5 [48]. Also, the Cu T1 in LACs is inserted in a hydrophobic pocket which allows favorable interaction with its natural organic substrates [49].…”

The Photocatalyzed Thiol‐ene reaction: A New Tag to Yield Fast, Selective and reversible Paramagnetic Tagging of Proteins

“…Being easily obtained, and in high yields, the formation of non‐reducible thioether bonds is convenient and makes this strategy a method of choice in the field of paramagnetic tagging, opening the way to development of more novel and rigid paramagnetic tags. Those tags could be applied to the study of larger systems, where the PCSs analysis could be complemented by the use of residual dipolar couplings and paramagnetic relaxation enhancements measurements …”

NMR Spectroscopy and Metal Ions in Life Sciences

“…For instance, in the complex formed by cytochrome P450cam and putidaredoxin, neither the crystal nor the solution structures are in agreement with the PRE measured by alternatively attaching a lanthanide binding tag, with a coordinated gadolinium(III) ion, in several positions along the protein chain . PRE could then be used to identify minor conformations, which can represent productive encounter states, connected to the main state through “electrostatic runways“, thus contributing to the association rate of the protein complex …”

“…PCS and RDC are particularly useful in the description of the interdomain conformational variability because, since they depend on a unique magnetic susceptibility anisotropy tensor, the data measured for the nuclei in the domain bearing the paramagnetic metal provides the tensor, which can then be used to recover information on the possible positions of the other domains from the averaged data measured for their nuclei . On the other hand, PRE are extremely useful to detect the presence of even lowly populated conformations where the detected nuclei are close to the paramagnetic metal , . The use of paramagnetic metals coordinated into proteins as reporters of their internal structural variability has thus become very popular.…”