  We present and analyze video-microscopy-based single-particle-tracking
measurements of the budding yeast (Saccharomyces cerevisiae) membrane protein,
Pma1, fluorescently-labeled either by direct fusion to the switchable
fluorescent protein, mEos3.2, or by a novel, light-touch, labeling scheme, in
which a 5 amino acid tag is directly fused to the C-terminus of Pma1, which
then binds mEos3.2. The diffusivity distributions of these two populations of
single particle tracks differ significantly, demonstrating that labeling method
can be an important determinant of diffusive behavior. We also applied
perturbation expectation maximization (pEMv2) [Physical Review E 94, 052412
(2016)], which sorts trajectories into the statistically-optimum number of
diffusive states. For both TRAP-labeled Pma1 and Pma1-mEos3.2, pEMv2 sorts the
tracks into two diffusive states: an essentially immobile state and a more
mobile state. However, the mobile fraction of Pma1-mEos3.2 tracks is much
smaller (0.1) than the mobile fraction of TRAP-labeled Pma1 tracks (0.5). In
addition, the diffusivity of Pma1-mEos3.2's mobile state is several times
smaller than the diffusivity of TRAP-labeled Pma1's mobile state. To critically
assess pEMv2's performance, we compare the diffusivity and covariance
distributions of the experimental pEMv2-sorted populations to corresponding
theoretical distributions, assuming that Pma1 displacements realize a Gaussian
random process. The experiment-theory comparisons for both the TRAP-labeled
Pma1 and Pma1-mEos3.2 reveal good agreement, bolstering the pEMv2 approach.
