DNA substrates will be labelled at precise locations along the DNA sequence with suitable (secondary) fluorophores capable of producing FRET signals with another fluorophore.  The effect of random fluorophore labelling of the motor protein will be determined and this system will be used to study the timing of FRET signals after addition of ATP using a  fluorescence stopped-flow device, by rapid mixing of ATP and protein-DNA solutions, as well as a microspectrofluorimeter, coupled to a flash photolysis system, which triggers DNA movement by photoactivation of caged ATP bound to the motor protein.  The accuracy with which the FRET signals from differently located secondary fluorophores can be detected, will be measured under a variety of conditions (e.g. temperature variation, buffer variation etc.). 

Outcomes:

Ports provided Parma with HsdR(prrI) protein and demonstrated that this can be labelled at the N-terminal Cys4 residue with a simple maleimide chemistry and Alexa633 fluorophore.  We have also provided two oligonucleotides, with and without 5’BHQ quenchers, that have been shown to FRET and quench Alexa633 fluorescence (using both gel technology and the COSMIC facility at Edinburgh University).

Parma has also provided evidence of FRET with HsdR(prrI) protein, both in the bulk and at the single molecule level. This was, however, better achieved using ATTO630 succinimide, instead of Alexa633 maleimide, because labelling on Lys residues was more efficient than on the unique Cys residue of Prr. ATTO derivatives also appear to be more stable photophysically than Alexa633 derivatives. Furthermore, BHQ3 30mer, instead of BHQ2, was used as acceptor, because more efficient as quencher for that labels. FRET was, however, found to be due to both specific and unspecific effects, which must be taken in proper account for future work development.

Parma provided evidence of FRET from HsdR(prrI) protein to DNA model systems, both at the ensemble and single molecule levels, using ATTO635 labelling of HsdR(PrrI) and a short (30mer) DNA oligomer labelled with either black hole quencher dye (BHQ3) or ATTO700. However, due to severe instrumental limitations (i.e. large detector cross-talks) a different instrumental setup and donor/acceptor couple were realized. Also, in order to synchronize start of translocation with fluorescence bursts acquisition under single molecule regime, a sub-ms flash photolysis system was exploited to convert caged ATP into active ATP. Thanks to the six months extension period, our confocal microscope was implemented with a second ps pulsed blue laser to exploit a very new technique, called PIE (pulsed interleaved excitation), that makes FRET detection much more reliable. As a consequence the new D/A couple ATTO488/ATTO633 was chosen to label motor protein and DNA fragments. FRET was then detected with model systems under static conditions, but FRET detection during translocation is still under way, due to the recent assembly of the new instrumental setup. We are still actively engaged to exploit PIE, to see if stronger FRET signals can be detected during DNA movement before the end of the project.