Protein self-association in crowded protein solutions: a time-resolved fluorescence polarization study.
The self-association equilibrium of a tracer protein, apomyoglobin (apoMb), in highly concentrated crowded solutions of ribonuclease A (RNase A) and human serum albumin (HSA), has been studied as a model system of protein interactions that occur in crowded macromolecular environments. The rotational diffusion of the tracer protein labeled with two different fluorescent dyes, 8-anilinonaphthalene-1-sulfonate and fluorescein isothiocyanate, was successfully recorded as a function of the two crowder concentrations in the 50-200 mg/mL range, using picosecond-resolved fluorescence anisotropy methods. It was found that apoMb molecules self-associate at high RNase A concentration to yield a flexible dimer. The apparent dimerization constant, which increases with RNase A concentration, could also be estimated from the fractional contribution of monomeric and dimeric species to the total fluorescence anisotropy of the samples. In contrast, an equivalent mass concentration of HSA does not result in tracer dimerization. This different effect of RNase A and HSA is much larger than that predicted from simple models based only on the free volume available to apoMb, indicating that additional, nonspecific interactions between tracer and crowder should come into play. The time-resolved fluorescence polarization methods described here are expected to be of general applicability to the detection and quantification of crowding effects in a variety of macromolecules of biological relevance.
Zorrilla
Silvia
S
Instituto de Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.
Rivas
Germán
G
Acuña
A Ulises
AU
Lillo
M Pilar
MP
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D004058:Diffusion; D019281:Dimerization; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes; D006736:Horses; D006801:Humans; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012259:Ribonuclease, Pancreatic; D012709:Serum Albumin; D012996:Solutions
D001059:Apoproteins; D005456:Fluorescent Dyes; D009211:Myoglobin; D011506:Proteins; D012709:Serum Albumin; D012996:Solutions; C010857:apomyoglobin; D012259:Ribonuclease, Pancreatic