Time-resolved phosphorescence anisotropy and photobleaching recovery characterization of protein aggregation induced by paraformaldehyde fixation of cells

Abstract

Antigen presentation by MHC class II molecules can be enhanced by paraformaldehyde fixation of antigen-presenting cells prior to assay. This treatment might be expected to aggregate membrane proteins and thus stabilize and strengthen transient protein-protein interactions involved in intercellular cooperation. Rotational and lateral dynamics of the MHC class II antigen I-A d on A20 cells fixed with various concentrations of paraformaldehyde were examined by time- resolved phosphorescence anisotropy and fluorescence photobleaching recovery, respectively. Probes were erythrosin and tetramethylrhodamine conjugates of MKD6 Fab fragments. Increasing concentrations of paraformaldehyde progressively increased I-A d 's limiting anisotropy at 4 degrees Celsius above the value of 0.042 seen in untreated cells while leaving the rotational correlation time of 22 microsecond unchanged. On the other hand, translational diffusion coefficients decreased from about 2 X 10 -10 cm 2 sec -1 while recovery remained unchanged at 40 - 50%. Together these results suggest that fixation crosslinks class II molecules with each other or with other membrane proteins into structures large enough (greater than 500,000 kDa) to appear rotationally immobile but small enough to diffuse translationally with size-dependent rates. Fixation effects on both class II rotation and lateral diffusion are half-maximal at paraformaldehyde concentrations of approximately 0.2%. Possible relations between biology of class II effector functions and physical sizes of fixation-induced aggregates are discussed.