Protein self‐association in crowded protein solutions: A time‐resolved fluorescence polarization study

Abstract

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.

Analysis of the size dependence of macromolecular crowding shows that smaller is better

“…For example, 200 mg/mL of dextran increases the stability of protein L by less than 0.5 kcal/mol (33), which is almost below the resolution of stability measurements. ApoMB dimerization was enhanced by 200 mg/mL of RNaseA but not by the same concentration of human serum albumin (34). Other examples are not rare (35,36) and perhaps are underreported in the literature as negative results often are.…”

Protein–protein interactions in a crowded environment

“…Thus, to answer the above questions, many experimental studies have been performed using synthetic polymers or specific proteins as crowding agents. These studies have addressed both the thermodynamics and kinetics of protein-protein interactions in a crowded environment (Minton and Wilf 1981;Minton 1983;Jarvis and Ring 1990;van den Berg et al 1999;Wenner and Bloomfield 1999;Morar et al 2001;Patel et al 2002;Kozer and Schreiber 2004;Zorrilla et al 2004;Phillip et al 2009;Wang et al 2011;Fodeke and Minton 2011). Using "inert" crowding agents, the primary focus in most of these studies was to understand the excluded volume effects of crowding agents on the formation of protein complexes (Minton 1983;Kim et al 2010).…”

Translational and rotational motions of proteins in a protein crowded environment

“…Among other biophysical techniques, methods based on fluorescence spectroscopy [11][12][13] appear to be very convenient for interaction studies in crowded media due to the possibility of specifically labeling only the tracer protein with extrinsic fluorescent dyes, which in this way can be distinguished easily from the crowding macromolecules. These techniques allow characterization and quantification of the hydrodynamic properties of free and bound species, both in crowded model solutions [14,15] and in whole living cells [6,[16][17][18], using their fluorescence. Time-resolved fluorescence depolarization (TRFD) measurements provide a detailed description of the rotationally depolarizing motions of the tracer molecules that take place on the nanosecond time scale.…”