Abstract
In the living cell, biomolecules perform their respective functions in the presence of not only one type of macromolecules but rather in the presence of various macromolecules with different shapes and sizes. In this study, we have investigated the effects of five single macromolecular crowding agents, Dextran 6, Dextran 40, Dextran 70, Ficoll 70, and PEG 8000 and their binary mixtures on the modulation in the domain separation of human serum albumin using a Förster resonance energy transfer-based approach and the translational mobility of a small fluorescent probe fluorescein isothiocyanate (FITC) using fluorescence correlation spectroscopy (FCS). Our observations suggest that mixed crowding induces greater cooperativity in the domain movement as compared to the components of the mixtures. Thermodynamic analyses of the same provide evidence of crossovers from enthalpy-based interactions to effects dominated by hard-sphere potential. When compared with those obtained for individual crowders, both domain movements and FITC diffusion studies show significant deviations from ideality, with an ideal solution being considered to be that arising from the sum of the contributions of those obtained in the presence of individual crowding agents. Considering the fact that domain movements are local (on the order of a few angstroms) in nature while translational movements span much larger lengthscales, our results imply that the observed deviation from simple additivity exists at several possible levels or lengthscales in such mixtures. Moreover, the nature and the type of deviation not only depend on the identities of the components of the crowder mixtures but are also influenced by the particular face of the serum protein (either the domain I–II or the domain II–III face) that the crowders interact with, thus providing further insights into the possible existence of microheterogeneities in such solutions.
Highlights
Biochemical studies of macromolecules are often done in dilute solutions where the macromolecular concentration is in the range of 1−10 g/L.1−4 These dilute environments differ dramatically from the interiors of cells or extracellular matrices of tissues and cartilages where the biological macromolecules are known to function.[4−6] Real biological environments contain a high density of macromolecular solutes as a part of the same medium where the test protein locates.[7,8] Depending upon the specific organelle, the total occupancy by macromolecules can be in the range of 5−40% of the available volume, corresponding to 50− 400 g/L of the total macromolecular concentration
We have studied a total of seven binary mixtures of some commonly used macromolecular crowders, namely, Dextran 6 in Dextran 40, Dextran 6 in Dextran 70, Dextran 6 in Ficoll 70, Dextran 6 in polyethylene glycol 8000 (PEG 8), Dextran 40 in Dextran 70, Dextran 40 in Ficoll 70, and Dextran 70 in Ficoll 70, thereby encompassing a range of shapes and sizes along with differences in the structure of the crowders at the molecular level
The choice of PEG 8000, which unlike the aforementioned ones, is not an ideal inert crowding agent as it is known to interact with proteins, was because of its molecular weight being similar to that of Dextran 6 and because of the fact that it is a linear open-chain polymer as opposed to the others that are typically constrained to a certain extent by the cyclic ring systems of the component sugar moieties
Summary
Biochemical studies of macromolecules are often done in dilute solutions where the macromolecular concentration is in the range of 1−10 g/L.1−4 These dilute environments differ dramatically from the interiors of cells or extracellular matrices of tissues and cartilages where the biological macromolecules are known to function.[4−6] Real biological environments contain a high density of macromolecular solutes (proteins, nucleic acids, polysaccharides, etc.) as a part of the same medium where the test protein locates.[7,8] Depending upon the specific organelle, the total occupancy by macromolecules can be in the range of 5−40% of the available volume, corresponding to 50− 400 g/L of the total macromolecular concentration. Macromolecular crowding has been explained based on the “excluded volume” effect[1−6] arising from the mutual impenetrability of the involved species, the latter being treated as hard spheres In this regard, a multitude of processes such as the protein folding−unfolding reaction,[9−11] protein−protein association,[12−14] protein aggregation,[15,16] to name a few, have been shown to be appreciably affected by the presence of macromolecular crowding agents. Recent papers have shown that the structure of HSA is significantly modulated in the presence of crowding agents.[46−48] Keeping in mind the fact that the serum protein undergoes large scale domain movements (angular displacements) when binding to FAs49,50 and exhibits allostery on ligand binding,[51,52] understanding and trying to comprehend the manner in which such displacements are affected in a mixed crowding scenario is of immense importance. With domain movements providing insights into the crowder arrangement in the immediate vicinity of the protein, this in combination with the translation diffusion studies suggest that nonideality exists over several orders of length scales, arguably from angstroms to nanometers, thereby further highlighting the importance of a mixed crowding scenario and its underlying complexity
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