<em>In situ</em> monitoring of specific and functional protein-protein interactions between redox proteins — ASN Events
  1. Schedule
  2. Session Ten - Poster Session C
  3. In situ monitoring of specific and functional protein-protein interactions between redox proteins

In situ monitoring of specific and functional protein-protein interactions between redox proteins (#330)

Joyee C.I. Yeung 1 , Stephen G. Bell 2 , Luet L. Wong 3 , Raymond J. Rodgers 4 , Lisandra L. Martin 1
  1. School of Chemistry, Monash University, Clayton, VIC, Australia
  2. School of Chemistry & Physics, The University of Adelaide, Adelaide, SA, Australia
  3. Department of Chemistry, University of Oxford, Oxford, OX1 3QR, UK
  4. Department of Obstetrics and Gynecology, The University of Adelaide, Adelaide, SA, Australia

Dynamic conformational changes are often correlated to protein function.A change in environmental factors (temperature,pH and shear stress) or the intrinsic properties of protein (redox state and chemical activation) can induce changes of protein conformation1.Our aim is to examine the structure-function relationship of redox active enzymes in order to gain an insight into conformational changes for reduced vs oxidised forms of partner proteins.We have selected a bacterial P450 enzyme (CYP199A4) that requires electrons to oxidatively demethylate 4-methoxybenzoic acid.CYP199A4 utilises a class I electron transfer system to transfer two electrons from the electron donor (NADH) to the P450 enzyme,one at a time,via two redox partner partner proteins,a ferredoxin reductase (HaPuR) and a ferredoxin (HaPux)2.We have studied protein-protein interactions using Quartz Crystal Microbalance (QCM) and Atomic Force Microscopy (AFM).The QCM detects a change in protein binding events (mass adsorbed) and the associated structural properties by measuring a change in frequency and energy dissipation, respectively3.We have used the QCM and AFM to monitor the structural changes of CYP199A4, with and without,the substrate,4-methoxybenzoic acid.These results showed no overall conformational changes,regardless of the presence of substrate.Secondly,we have examined the interaction of three ferredoxins (the electron transfer shuttle) with CYP199A4.Finally,the QCM measurements reveal significant conformation changes to the CYP199A4:ferredoxin complexes when NADH and ferredoxin reductase (HaPuR) are introduced.The reduced HaPuR binds more tightly to the CYP199A4:ferredoxin complexes compared with oxidized HaPuR.In addition, this binding was enhanced under reduced (low) ionic strength conditions suggesting that the charged protein surfacess are less shielded by ions, promoting stronger protein-protein interactions.The ability to modify the redox properties of HaPuR,results in an altered conformation that appears to enhance binding to the CYP199A4-ferredoxin complexes.The use of QCM to create of an in vitro environment in which the intrinsic properties of enzymes with their partner proteins can be studied,offers numerous potential applications for biocatalysis. 

  1. J. H. Ha, S. N. Loh, Chemistry: A European Journal 2012, 18, 7965 - 8243
  2. S. G. Bell, W. Yang, A. B. H. Tan, R. Zhou, E. O. D. Johnson, A. Zhang, W. Zhou, Z. Rao, L.-L. Wong, Daltons Transactions 2012, 41, 8703 - 8714
  3. S. Praporski, S. M. Ng, A. D. Nguyen, C. J. Corbin, A. Mechler, J. Zheng, A. J. Conley, L. L. Martin, The Journal of Biological Chemistry 2009, 284(48): 33224-33232.