Labeling, tagging and charging - Long standing issues in bioenergetics addressed with novel techniques

Sammanfattning: Understanding the molecular details of bioenergetic processes is fundamentally important. Defects in the mitochondrial energy metabolism can lead to untimely apoptosis and thus cause serious degenerative human diseases. Other defects in mitochondrial energy metabolism can have the reverse effect and cause cell immortality and also contribute to and high metastatic potential of tumor cells. Knowing the location of active sites in proteins is key to understanding their function. In paper V of this work we have produced novel photo-reactive quinone analogues and analyzed their ability to specifically label quinone binding sites in proteins upon illumination with long wavelength UV light. Three different bioenergetic model enzymes of different complexity were used to test the labeling potential, both in situ in the natural membrane and in detergent solubilized and purified proteins. The reaction products formed after illumination was analyzed by mass spectrometry using model peptides as protein substrates. Fusion protein techniques are commonly used to add a “tag” to proteins, extending the natural protein with an extra domain, like a his-tag or green fluorescent protein, to be used for purification or detection purposes respectively. In paper II and III of this work we have added a cytochrome c domain to two different protein subunits of NADH:quinone oxidoreductase (respiratory chain Complex I). One of the major obstacles in Complex I research so far, has been the absence of visual redox pigments in the enzyme. The new cytochrome tag gives the proteins a red color, aid in purification and allows precise quantification of both individual subunits and of intact Complex I. With the help of the cytochrome tag, novel issues concerning the spatial organisation and functional mechanism of the membrane-spanning domain of Complex I can be addressed. Direct electrochemistry is a fruitful method to investigate the electronic events of bioenergetic processes in enzymes. In paper I we have used a spectro-electrochemical cell allowing us directly correlate spectral events and redox events, to investigate redox properties of succinate:quinone oxidoreductase (respiratory chain Complex II) in the presence and the absence of quinone mediators and a specific inhibitor. The measurements revealed previously unknown redox dependent conformational changes in the enzyme. Bioinformatics is a powerful technique to understand the evolution and function of proteins. If both the primary sequences and the three dimensional structures of the proteins can be compared, as we have done in paper IV with bacterial flavodoxins with eukaryotic NAD(P)H:quinone oxidoreductases, the molecular details of catalyses can be better understood and the functional diversification in a protein family revealed.

  Denna avhandling är EVENTUELLT nedladdningsbar som PDF. Kolla denna länk för att se om den går att ladda ner.