Molecular probes as protein analysis tools in biotechnology

Abstract: Protein-labeling today is a work of art, in vivo studies of proteins or other molecules can easily be performed, and the movement of the labeled molecule can be followed in real time. Labeling in vitro gives enormous amount of data in labs all over the world on a daily basis, where protein-protein, protein-DNA or other interactions are studied. Folding and unfolding events can be monitored wi th labels reporting on local or global environmental changes in a protein. The use of labeling seems endless, but in this thesis I have chosen to focus on two labeling techniques: spin-labeling and fluorescence labeling. Applying these techniques on protein-protein and protein-DNA interactions has resulted in better understanding of protein folding and function.Chaperonin function at elevated temperaturesThe model protein HCA II (259 amino acids) mainly consisting of a large 10 stranded ß-sheet with a topological breakpoint between strands 6 and 7. Two residues, adjacent in the folded structure and located at each side of this breakpoint, were used in a site-directed-spin-labeling (SDSL) experiment. The aim was to elucidate what happens at the breakpoint when the protein interacts with the chaperonin GroEL at elevated temperatures. The chaperonin GroEL is a 60 kDa protein known to aid protein folding in the cell. By probing the model protein, HCA II, we have shown that this chaperone can stretch its substrate and release it for a new refolding opportunity.MexR protein interaction with DNAMexR is a 147 amino acid protein dimer involved in transcriptional repression of the multidrug efflux operon MexAB-OprM in the opportunistic bacterial pathogen Pseudomonas aeruginosa. Malfunction in MexR results in multidrug resistant bacteria resistant to therapeutic strategies. Site-specific fluorescence-labeling of MexR has been investigated as a means to provide a new strategy for localising DNA binding and quantifyi ng DNA affinity. ANS fluorescence of the MexR protein in the absence and presence of DNA, together with a range of biophysical measurements, has provided a new view on how MexR could be regulated by small molecule binding, and thus sheds new light on its functionality in gene repression.