Quantitative Investigation of Biomolecular Interactions in Crowded Media by Fluorescence Spectroscopy, a Good Choice

Abstract

Fluorescence spectroscopy methods have been proved to be powerful tools for the quantitative investigation of homologous and heterologous interactions, rotational and translational diffusion, and structural dynamics of biological molecules in crowded media. In addition to their high sensitivity, these methods present the advantage that the selective fluorescent labeling of the biomolecules under study allows distinguishing them from the background species. Moreover, the recent development of biological applications of single molecule fluorescence micro-spectroscopy methods has opened the possibility of performing quantitative determinations inside cells. In the last decades, theoretical and experimental studies have demonstrated the possible influence of the high concentration of macromolecules within living systems, on the thermodynamics and kinetics of biological reactions. Therefore, there is a growing interest in quantitatively evaluating the interactions involving biomolecules in the natural environment in which they occur. Since this is not always feasible, experiments conducted in model crowded conditions, resembling physiological media, may contribute to reduce the gap between traditional in vitro and in vivo experiments. In this review we will discuss the application of some fluorescence spectroscopy approaches, for the identification and quantification of biological macromolecules and their functional interactions in model crowded conditions.

Impact of Molecular Crowding on Translational Mobility and Conformational Properties of Biological Macromolecules

“…Although the obtained crowded solutions (in vitro studies) do not completely mimic all properties of the cytosolic environment (in vivo studies), they provide a wellcontrollable environment, because parameters like concentration, size, shape, diffusivity, and composition of crowding agents can be varied systematically. 1,11,19 In this work, we investigated the translational diffusion and the conformational changes of various biological macromolecules as a function of different crowding conditions. These properties represent crucial parameters for almost any cellular process and by this regulate activities in living cells.…”

“…(i) They should be inert, resulting in minimal chemical interactions with the target molecule, and (ii) they should be highly soluble in aqueous buffers. Although the obtained crowded solutions (in vitro studies) do not completely mimic all properties of the cytosolic environment (in vivo studies), they provide a well-controllable environment, because parameters like concentration, size, shape, diffusivity, and composition of crowding agents can be varied systematically. ,, …”

The Shape of Protein Crowders is a Major Determinant of Protein Diffusion

“…To mimic physiological media in in vitro experiments, a total concentration of macromolecules of up to 400 g/L is commonly used (7,11). Frequently, random-coil polymers, such as polyethylene glycol, Ficoll, or dextran, are used as macromolecular crowding agents because of their ease of availability and experimental manipulation (12,13). However, these nonbiological crowding agents may not reveal physiologically relevant information (2,3).…”

“…Because the measured correlation function reflects the kinetics of molecules diffusing in and out of the detection volume of a confocal microscope, it is an ideal analytical tool for studying molecular dynamics (20,21), concentration (15), chemical reaction kinetics (22), and the interactions of molecules at nanomolar concentrations (15,(23)(24)(25). In the last decade, FCS has become a standard technique for studying diffusion, including anomalous diffusion and biomolecular interactions in crowded environments (12,(26)(27)(28)(29).…”

Hindered Diffusion in Polymeric Solutions Studied by Fluorescence Correlation Spectroscopy

“…An alternative, noninvasive technique to quantitatively investigate diffusion is fluorescence correlation spectroscopy (FCS) (7). FCS has been used in a variety of systems to estimate diffusion of molecules in crowded media (8), and to examine various molecular interactions (9)(10)(11), including binding (12). Rusu et al (13) used the amplitude of the autocorrelation function to study the binding of proteins to phospholipid vesicles.…”