Protein Phosphorylation and Intermolecular Electron Transfer:  A Joint Experimental and Computational Study of a Hormone Biosynthesis Pathway

Zöllner, Andy; Pasquinelli, Melissa A.; Bernhardt, Rita; Beratan, David N.

doi:10.1021/ja064803j

Cited by 22 publications

(18 citation statements)

References 84 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All samples were prepared in a glove box in an oxygen‐free atmosphere. The reaction buffer applied for all measurements was a 50 m m Hepes buffer (pH 7.4) containing 0.05% Tween 20 [39].…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Purification and functional characterization of human 11β hydroxylase expressed in Escherichia coli

et al. 2008

Self Cite

View full text Add to dashboard Cite

The final steps in the synthesis of the major human glucocorticoid, cortisol, and the most important mineralocorticoid in humans, aldosterone [1], are catalyzed by 95% identical mitochondrial cytochrome P450 isozymes, 11b-hydroxylase (CYP11B1; EC 1.14.15.4) and CYP11B2 [2]. Cortisol is synthesized from 11-deoxycortisol through a hydroxylation reaction at position 11b catalyzed by CYP11B1, whereas aldosterone is synthesized from 11-deoxycorticosterone via a series of reactions catalyzed by CYP11B2.CYP11B1 is expressed in the adrenal zona fasiculata and is regulated by adrenocorticotrophic hormone (ACTH). The human aldosterone synthase, CYP11B2, on the other hand, is expressed in the zona glomerulosa The human 11b-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-containing reductase. On the biochemical and biophysical level, little is known about hCYP11B1 because it is very unstable for analyses performed in vitro. This instability is also the reason why it has not been possible to stably express it so far in Escherichia coli and subsequently purify it. In the present study, we report on the successful and reproducible purification of recombinant hCYP11B1 coexpressed with molecular chaperones GroES ⁄ GroEL in E. coli. The protein was highly purified to apparent homogeneity, as observed by SDS ⁄ PAGE. Upon mass spectrometry, the mass-tocharge ratio (m ⁄ z) of the protein was estimated to be 55 761, which is consistent with the value 55 760.76 calculated for the form lacking the translational initiator Met. The functionality of hCYP11B1 was analyzed using different methods (substrate conversion assays, stopped-flow, Biacore). The results clearly demonstrate that the enzyme is capable of hydroxylating its substrates at position 11-beta. Moreover, the determined NADPH coupling percentage for the hCYP11B1 catalyzed reactions using either 11-deoxycortisol or 11-deoxycorticosterone as substrates was approximately 75% in both cases. Biacore and stopped-flow measurements indicate that hCYP11B1 possesses more than one binding site for its redox partner adrenodoxin, possibly resulting in the formation of more than one productive complexes. In addition, we performed CD measurements to obtain information about the structure of hCYP11B1.Abbreviations ACTH, adrenocorticotrophic hormone; AdR, adrenodoxin reductase; Adx, adrenodoxin; bCYP11B1, bovine 11b hydroxylase; hCYP11B1, human 11b hydroxylase.

show abstract

Section: Methodsmentioning

confidence: 99%

“…Formation of the bAdx ⁄ bCYP11B1 and bAdx ⁄ hCYP11B1 complexes was assayed on a Biacore 3000 system (Biacore, Uppsala, Sweden), using the optical biosensor method described previously [39] with slight modifications.…”

Section: Optical Biosensor Measurementsmentioning

confidence: 99%

Purification and functional characterization of human 11β hydroxylase expressed in Escherichia coli

et al. 2008

Self Cite

View full text Add to dashboard Cite

show abstract

“…Brownian dynamics (BD) has been widely used in recent years to study the protein association process (34)(35)(36)(37)(38)(39)(40). In most BD simulations, proteins are treated as rigid bodies and are moved by the Brownian forces stochastically (41).…”

Section: Introductionmentioning

confidence: 99%

Brownian Dynamics and Molecular Dynamics Study of the Association between Hydrogenase and Ferredoxin from Chlamydomonas reinhardtii

Long

Chang

King

et al. 2008

Biophysical Journal

View full text Add to dashboard Cite

The [FeFe] hydrogenase from the green alga Chlamydomonas reinhardtii can catalyze the reduction of protons to hydrogen gas using electrons supplied from photosystem I and transferred via ferredoxin. To better understand the association of the hydrogenase and the ferredoxin, we have simulated the process over multiple timescales. A Brownian dynamics simulation method gave an initial thorough sampling of the rigid-body translational and rotational phase spaces, and the resulting trajectories were used to compute the occupancy and free-energy landscapes. Several important hydrogenase-ferredoxin encounter complexes were identified from this analysis, which were then individually simulated using atomistic molecular dynamics to provide more details of the hydrogenase and ferredoxin interaction. The ferredoxin appeared to form reasonable complexes with the hydrogenase in multiple orientations, some of which were good candidates for inclusion in a transition state ensemble of configurations for electron transfer.

show abstract

“…A list of 308 substrates reported in 2003, including transcription factors, signaling proteins, effectors of DNA/ RNA structure and function, structural proteins, metabolic enzymes, etc., certainly underestimates the real number (98). Many new CK2 substrates have been added, e.g., components of the steroid hormone cascade, capable of modulating the biosynthesis of steroid hormones (99,100).…”

Section: Protein Kinasesmentioning

confidence: 99%

Interactions of natural polyamines with mammalian proteins

Schuster

Bernhardt

2011

BioMolecular Concepts

Self Cite

View full text Add to dashboard Cite

The ubiquitously expressed natural polyamines putrescine, spermidine, and spermine are small, flexible cationic compounds that exert pleiotropic actions on various regulatory systems and, accordingly, are essentially involved in diverse life functions. These roles of polyamines result from their capability to interact with negatively charged regions of all major classes of biomolecules, which might act in response by changing their structures and functions. The present review deals with polyamine-protein interactions, thereby focusing on mammalian proteins. We discuss the various modes in which polyamines can interact with proteins, describe major types of affected functions illustrated by representative examples of involved proteins, and support information with respective structural evidence from elucidated three-dimensional structures. A specific focus is put on polyamine interactions at protein surfaces that can modulate the aggregation of proteins to organized structural networks as well as to toxic aggregates and, moreover, can play a role in important transient protein-protein interactions.

show abstract

Protein Phosphorylation and Intermolecular Electron Transfer: A Joint Experimental and Computational Study of a Hormone Biosynthesis Pathway

Cited by 22 publications

References 84 publications

Purification and functional characterization of human 11β hydroxylase expressed in Escherichia coli

Purification and functional characterization of human 11β hydroxylase expressed in Escherichia coli

Brownian Dynamics and Molecular Dynamics Study of the Association between Hydrogenase and Ferredoxin from Chlamydomonas reinhardtii

Interactions of natural polyamines with mammalian proteins

Contact Info

Product

Resources

About