In recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra, correlation spectroscopy and anisotropy decay were used to characterize labelling and to obtain photophysical parameters of free and peptide-bound FlAsH. The data demonstrates that FlAsH is a viable probe for single-molecule studies. Single-molecule imaging confirmed dual labeling of the peptide with FlAsH and AF647. Multiparameter single-molecule Förster Resonance Energy Transfer (smFRET) measurements were performed on freely diffusing peptides in solution. The smFRET histogram showed different peaks corresponding to different backbone and dye orientations, in agreement with the molecular dynamics simulations. The tandem of fluorophores and the labelling strategy described here are a promising alternative to bulky fusion fluorescent proteins for smFRET and single-molecule tracking studies of membrane proteins.

ATP dependent molecular machines of the AAAþ family perform many cellular activities and are grouped into two classes depending on if they have one or two ATPase domains or ''rings''. E. coli ClpA, a AAAþ protein unfoldase and part of the ClpAP protease is one of the best-characterized enzymes belonging to the two-ATPase domain class. The ClpA ATPase domains are referred to as the D1 and D2 domains, with predominant ATP turnover performed by D2. In the current study, we have studied a Walker B mutant, E286Q in the ATPase active site of the D1 domain that abolishes ATP hydrolysis but doesn't impair ATP binding. Our single-molecule optical trap experiments have dissected the effect of the non-hydrolyzing D1 domain on unfolding and translocation of substrates by ClpAP. Although in the wildtype enzyme the D1 domain hydrolyzes only $10% of total ATP turned over by ClpAP, abolishing this hydrolysis reduced the unfolding rate of the model substrate, titin I27 V13P-ssrA from the C-terminal end, $ 3-fold and the average translocation velocity by $25%. The D1 mutant had a greater effect from the N-terminus as shown by studies with a related substrate, ssrA-I27 V15P ; this substrate was unfolded $10 times slower and the average translocation velocities were reduced by $60%. Slower translocation was primarily caused by an increased frequency of pausing and longer pausing times compared to the wild-type enzyme. These results suggest that defects in ATP-hydrolysis in the D1 ring cause loss of coordination between the two rings resulting in misfiring of the D2 motor domain. Thus, our single-molecule studies provide new insight into the functional importance of the ClpA D1 ATPase domain and the role of coordination between each ClpA AAAþ ring, which were not apparent in bulk biochemical analyses.