Redox‐dependent dynamics of putidaredoxin characterized by amide proton exchange

Lyons, Teresa A.; Ratnaswamy, Gayathri; Pochapsky, Thomas C.

doi:10.1002/pro.5560050407

Cited by 54 publications

(67 citation statements)

References 54 publications

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“…All NMR data were processed in NmrPipe (55) and analyzed in CCPNMR (56). The amide resonances of oxidized Pdx and cytP450cam were assigned based on previous works (24,57).…”

Section: Methodsmentioning

confidence: 99%

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

Andrałojć

Hiruma

Wei-min

et al. 2017

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

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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...

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“…All NMR data were processed in NmrPipe (55) and analyzed in CCPNMR (56). The amide resonances of oxidized Pdx and cytP450cam were assigned based on previous works (24,57).…”

Section: Methodsmentioning

confidence: 99%

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

Andrałojć

Hiruma

Wei-min

et al. 2017

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

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“…Several other examples exist that utilize a similar approach and reach the same conclusion. [180][181][182] The conformational stability and biological activity of Calmodulin (CaM) before and after methionine oxidation provides another example of the relationships between protein flexibility and oxidation. 183 The rate of methionine oxidation was shown to correlate with solvent exposure based on mass spectrometry measurements and resolution of different forms of oxidized CaM.…”

Section: Oxidationmentioning

confidence: 99%

The Complex Inter-Relationships Between Protein Flexibility and Stability

Kamerzell¹,

Middaugh²

2008

Journal of Pharmaceutical Sciences

108

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“…In addition, redox-active proteins often show different dynamics depending upon oxidation state, and redox-dependent dynamics can have functional consequences. For example, the Fe 2 S 2 ferredoxin putidaredoxin (Pdx) is both the physiological reductant and allosteric effector for CYP101, and shows structural and dynamic changes as a function of oxidation state which regulates its binding to CYP101 [4,5]. We wished to determine whether CYP101 also displays redox-dependent dynamics, and chose to use mass spectrometry (MS) to measure hydrogen/deuterium (H/D) exchange of backbone amide hydrogens as a probe of local backbone dynamics as a function of heme oxidation state.…”

Section: Introductionmentioning

confidence: 99%

Hydrogen–deuterium exchange mass spectrometry for investigation of backbone dynamics of oxidized and reduced cytochrome P450cam

Hamuro¹,

Molnar²,

Coales³

et al. 2008

Journal of Inorganic Biochemistry

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Backbone dynamics of the camphor monoxygenase cytochrome P450(cam) (CYP101) as a function of oxidation/ligation state of the heme iron were investigated via hydrogen/deuterium exchange (H/D exchange) as monitored by mass spectrometry. Main chain amide NH hydrogens can exchange readily with solvent and the rate of this exchange depends upon, among other things, dynamic fluctuations in local structural elements. A fluxional region of the polypeptide will exchange more quickly with solvent than one that is more constrained. In most regions of the enzyme, exchange rates were similar between oxidized high-spin camphor-bound and reduced camphor- and CO-bound CYP101 (CYP-S and CYP-S-CO, respectively). However, in regions of the protein that have previously been implicated in substrate access by structural and molecular dynamics investigations, the reduced enzyme shows significantly slower exchange rates than the oxidized CYP-S. This observation corresponds to increased flexibility of the oxidized enzyme relative to the reduced form. Structural features previously found to be perturbed in CYP-S-CO upon binding of the biologically relevant effector and reductant putidaredoxin (Pdx) as determined by nuclear magnetic resonance are also more protected from exchange in the reduced state. To our knowledge, this study represents the first experimental investigation of backbone dynamics within the P450 family using this methodology.

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Redox‐dependent dynamics of putidaredoxin characterized by amide proton exchange

Cited by 54 publications

References 54 publications

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

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

The Complex Inter-Relationships Between Protein Flexibility and Stability

Hydrogen–deuterium exchange mass spectrometry for investigation of backbone dynamics of oxidized and reduced cytochrome P450cam

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