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FRINATEK-Fri prosj.st. mat.,naturv.,tek

Structural basis for electron transfer in and between redox proteins - recognition, selectivity and flexibility.

Alternative title: Strukturelt grunnlag for elektronoverføringer i og mellom redoksproteiner - gjenkjenning, selektivitet og fleksibilitet.

Awarded: NOK 3.1 mill.

Project Number:

231669

Project Period:

2014 - 2018

Location:

Partner countries:

Proteins are essential components of all type of organisms. They have numerous different functions from catalysing biochemical reactions, sending and receiving signals to build up structural components. One type of these proteins is the redox proteins, and some of these are involved in electron transport. All organisms rely on electron transport to facilitate molecular transformations and for fundamental processes ranging from substrate oxidation to respiration and photosynthesis. Key to these roles is the formation of complexes between different proteins during electron transfer, which is predominantly performed by proteins containing so-called redox active cofactor. The structural basis for the control of specificity between these redox partner proteins has largely been lacking. The project has focused on addressing these issues, and to understand how these proteins recognise and associate with each other and how these redox networks have activated different enzyme systems. We have worked on mapping these interactions in an organism called Bacillus cereus by investigating these proteins with different biochemical and biophysical methods. This has given new insight with respect to selectivity, specificity and flexibility of these proteins. The sequential electron transfer occur here from NADPH to the flavodoxin reductases to the flavodoxins and finally to the redox enzyme. In Bacillus cereus we have expressed, purified and crystallised all three flavodoxin reductases, and solved the structure of two of them. For one of the pathways kinetic and binding studies have shown that the flavodoxin reductases can active the ribonucleotide reductase enzyme system, and that one of the reductases is the preferred redox partner. We have further studied the rest of this flavin redox network with a total of 6 proteins and 3x3 combinations of redoxpartners, and shown by kinetics that that for this network in Bacillus cereus, there is one set of redox partners that have a much higher activity than the other combinations. We have shown that a guide for this discrimination lays in the redox potentials, which we have determined for all the partners. Addiontally, we have identified a new subclass of the so-called thioredoxin-like flavodoxin reductases. Further, the structure of one of the flavodoxins have been solved. This has given new insight into the hydrogen bonding network around the flavin group, which is important in understanding the tuning of the redox activity of this protein. Due to the very high subatomic resolution of these structures we have been able to distinguish out that part of the flavin groups in these structures are actually disordered with a major and minor conformation. The structure of the flavodoxin reductase homolog thioredoxin reductase in this system has been determined. This has shown that only one of the NADPH sites are naturally occupied, which raises the question about negative cooperativity, which needs further investigations. For the other enzyme system we have studied, nitric oxide synthase, we have from binding studies gained new insight into the heme environment of the neuronal variant, and identified a new diflavin reductase for the bacterial variant. The results that have been describe above have been presented at many national and international conferences as the European crystallography meetings, the international bioinorganic chemistry conferences, he international flavin symposium, Gordon conference on enzymes and others both through posters and lectures. The results described above have resulted in several published articles in the journals Biochemistry (2x), Protein Science, Bio-Protocols, Febs Open Bio and Acta Crystallographica. The last results on e.g. nitric oxide synthase, high-resolution flavodoxin structures and thioredoxin reductase are being written together, and will be published in several publications. Additionally, we have published a paper in the Journal Biochemistry and Molecular Biology Education about a 2.5 week intensive course, which is based on our research from one of the flavodoxin-like proteins in the redox network studied in this project. It is important to increase the research relevance also in general bachelor- and master courses, which this course is an example of. The research results from the project are available for both national and international researchers, and can results in new studies within this field. The project has focused on the central goal of increasing the understanding of some redox networks in a model organism, to both characterise the individual proteins and the interaction between them. It has therefor been a balance between safer and more challenging subprojects. The research is basic research, but this fundamental understanding of enzyme activation and electron transfer can have relevance toward medical research for inhibition of enzymes, and toward industry which seek to optimise enzymes for industrial production.

Prosjektet har hatt fokus på å forstå hvordan biologiske systemer som involverer aktivering av redoksenzymer fungerer på et molekylært og atomært nivå ved å bruke en kombinasjon av forskjellige biokjemiske og biofysikalske metoder. Gjennom prosjektet har man fått økt forståelse av enzymaktiveringsnettverket for en gruppe redoksproteiner i bakterier, og vist at de enzymsystemene som har blitt studert har mer dedikerte aktiveringspartnere selv når det er flere muligheter. Videre har det vist at drivkraften i større grad er bestemt av elektronoverføringen enn bindingsinteraksjonen. Dette er kunnskap som kan brukes med tanke på både medisinsk og industriell anvendelse. Prosjektet har hatt internasjonalt samarbeid i skjæringspunktet mellom kjemi, biologi og fysikk samt benyttet avanserte internasjonale forskningsanlegg. Dette har fungert som en kompetanse og nettverksbygging for personene involvert i prosjektet.

Redox proteins acting in electron transfer are essential for life. All organisms rely on electron transport to facilitate molecular transformations and for fundamental processes ranging from substrate oxidation to respiration and photosynthesis. Key to th ese roles is the formation of transient inter-protein electron transfer complexes. The electron transfers are predominantly performed by proteins containing redox active cofactor as hemes, iron-sulfur clusters, flavins, disulfides, quinonens or NADPH. The structural basis for the control of specificity between these redox partner proteins is largely lacking. We will address these issues, and understand how these proteins recognise and associate with each other, and how specific, selective and flexible the se protein-protein interactions are. We have recently solved the structure of several of these proteins in Bacillus cereus, and also the protein-complex between a flavodoxin-like protein and a di-metal protein. We intend to try to map these interactions b y combining co-crystallisation of all the different complexes, and standard biochemical and biophysical methods to determine binding constants, redox potentials and kinetics, supplemented by spectroscopic studies (UV-vis, Raman and EPR spectroscopy). Some of these interactions are more unspecific, while others are very specific. The crystallisation of all of the protein-protein complexes will be challenging especially for those with very unspecific interactions. By mapping larger networks of redox protein s and enzyme systems, we hope to reveal more of the structural basis governing these interactions, which would be a clear step forward in understanding of electron transfer in proteins. This insight will further be linked to understanding the mechanism of the partner enzymes.

Publications from Cristin

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Funding scheme:

FRINATEK-Fri prosj.st. mat.,naturv.,tek