Effect of micro-viscosity on the rotational diffusion: pulsed laser-based time-resolved single-molecule study

The effect of the changes in buffer concentrations or any additives like surfactants in the protein samples during the analysis on the single-biomolecule diffusion is one of the hidden points in the single-molecule time-resolved measurements. In the current work, phosphoglycerate kinase (PGK) labeled with Atto-647 has been investigated on the single molecule level while it diffuses in Na2CO3 buffer at concentrations that vary from 10 mg/l up to 50 mg/l. The fluorescence lifetime of PGK labeled with Atto-647 in 50 mg/ml Na2CO3 has been measured, and it was found to be 2.7 ns. The fluorescence cross correlation of the diffused protein has also been measured, which confirms that the used samples are at a single molecule level. Time decay fluorescence anisotropy has been performed for PGK labeled with Atto-647 in different concentrations of Na2CO3, and the results confirmed that there is a clear impact on the molecular translational and rotational diffusion even with slight changes in the buffer concentration.