The effects of macromolecular crowding on the mechanical stability of protein molecules

Abstract

Macromolecular crowding, a common phenomenon in the cellular environments, can significantly affect the thermodynamic and kinetic properties of proteins. A single-molecule method based on atomic force microscopy (AFM) was used to investigate the effects of macromolecular crowding on the forces required to unfold individual protein molecules. It was found that the mechanical stability of ubiquitin molecules was enhanced by macromolecular crowding from added dextran molecules. The average unfolding force increased from 210 pN in the absence of dextran to 234 pN in the presence of 300 g/L dextran at a pulling speed of 0.25 mm/sec. A theoretical model, accounting for the effects of macromolecular crowding on the native and transition states of the protein molecule by applying the scaled-particle theory, was used to quantitatively explain the crowding-induced increase in the unfolding force. The experimental results and interpretation presented could have wide implications for the many proteins that experience mechanical stresses and perform mechanical functions in the crowded environment of the cell.

RETRACTED: Probing osmolyte participation in the unfolding transition state of a protein

“…Mechanical unfolding experiments of the protein I27 in aqueous glycerol solutions measured a considerable increase in the mechanical stability of the protein, with the average unfolding force increasing by approximately 50% (13). Similarly, in the presence of 30% dextran, a polysaccharide molecule, the average unfolding force of the protein ubiquitin increases by approximately 11% (35). Conversely, denaturing osmolytes such as guanidinium chloride have been shown to decrease the mechanical stability of the small protein GB1 (the B1 immunoglobulin-binding domain of protein G from Streptococcus) by approximately 80% in 2.25 M GdmCl compared with an aqueous solution (36).…”

The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding

“…The lower limit is due to the large mechanical loop of the AFM apparatus, which makes thermal drift the limiting factor. At high pulling speeds viscous drag becomes a problem [115][116][117][118]. At speeds above 10 µm/s, the speed-dependent hydrodynamic drag force acting on a cantilever is of the same magnitude as the unfolding force measured on single biological molecules [116].…”

Power-law dependence of the melting temperature of ubiquitin on the volume fraction of macromolecular crowders

“…[1][2][3] Thus, many in vitro experiments [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] and computer simulations 10,14,15,[21][22][23][24][25][26][27][28][29] have been carried out to determine, for example, the influence of macromolecular crowding on the energy landscape of protein folding and protein-protein interactions. Additionally, several models have been developed, aiming to provide a theoretical basis for understanding the effect of macromolecular crowding on various biological events.…”