Bulletin of the American Physical Society
APS March Meeting 2011
Volume 56, Number 1
Monday–Friday, March 21–25, 2011; Dallas, Texas
Session Q4: Macromolecular Crowding Effects in the Cytoplasm |
Hide Abstracts |
Sponsoring Units: DPOLY DBP Chair: Arun Yetheraj, University of Wisconsin Room: Ballroom A4 |
Wednesday, March 23, 2011 11:15AM - 11:51AM |
Q4.00001: Formation of protein-complexes in crowded environments: from in vitro to in vivo Invited Speaker: Rates of protein interactions are one to five orders of magnitude slower than the theoretically calculated collision rate of spheres of the same size. The rates can be increased by favorable electrostatic forces between the two proteins. Recent studies have established that the association reaction proceeds through transient complexes, which may be specific or diffusive in nature. To bring binding studies closer to the in vivo environment, we investigated the role of crowding on binding. For crowding we added various polymers to the solution, including Dextran and PEGs of different molecular weights. While crowding enhances oligomerization and polymerization of macromolecules, it has only a small effect on the binding rates and affinities of transient protein-protein interactions. We suggest that the limited effect of crowders, which is much bellow the expected from the increased viscosity of the solutions, is a result of the occluded volume effect in high crowder concentrations. Direct measurements of the stability of the encounter complex shows that crowders slow both k1 and k-1, resulting in an increased half-life of the encounter complex. High crowder concentrations also slow k2, suggesting an increased size of the encounter region. These results fit double-mutant cycle measurements on the activated complex, which suggest an increased size of the fruitful encounter region. These results are in line with the suggested occluded volume effect of crowders. We contrasted these with the effect of crowding on the weak binding pair CyPET-YPET. On this pair, aggregation, and not enhanced dimerization, was detected in PEG solutions. The results suggest that typical crowding agents have only a small effect on specific protein-protein dimerization reactions while promoting aggregation. To further validate these results, we performed real time binding assays in living cells, showing that even in the crowded cellular environment binding can be fast and specific. [Preview Abstract] |
Wednesday, March 23, 2011 11:51AM - 12:27PM |
Q4.00002: Crowding effects on protein association Invited Speaker: The cell cytoplasm is a dense environment where the presence of inert cosolutes can significantly alter the rates of protein folding and protein association reactions. These crowding effects can either increase or decrease the rates of association reactions (or protein folding) depending on the nature of the crowding agents and the type of reaction. Our work aims to obtain a quantitative understanding of crowding effects. We present the first kinetic study of the effect of hard sphere crowding agents on protein association reactions where reactants and crowding agents are both hard spheres. If every collision results in a reaction, crowding always decreases the reaction rate but if the probability of a reaction is low then crowding increases the reaction rate. We find that the thermodynamics of crowding are relatively insensitive to interactions between the crowding agents suggesting that the hard sphere model of crowding agents has a surprisingly large regime of validity, and should be sufficient for a qualitative understanding of the thermodynamics of crowding effects. [Preview Abstract] |
Wednesday, March 23, 2011 12:27PM - 1:03PM |
Q4.00003: Protein structure, stability and folding in the cell -- \textit{in silico} biophysical approaches Invited Speaker: How the crowded environment inside a cell affects the structural conformation of a protein with aspherical shape is a vital question because the geometry of proteins and protein-protein complexes are far from globules in vivo. Here we address this question by combining computational and experimental studies of several aspherical proteins (calmodulin, VlsE, and phosphoglycerate kinase) under crowded, cell-like conditions. The results show that macromolecular crowding affects protein folding dynamics, structures and functions. Our work demonstrates the malleability of ``native'' proteins and implies that crowding-induced shape changes may be important for protein function and malfunction in vivo. [Preview Abstract] |
Wednesday, March 23, 2011 1:03PM - 1:39PM |
Q4.00004: Phosphoglycerate kinase in crowded and cellular environments Invited Speaker: We developed the temperature-jump fluorescence microscope to spatio-temporally resolve fast biomolecular kinetics and stability inside a single mammalian cell. We measured the reversible fast folding kinetics as well as folding thermodynamics of a fluorescent phosphoglycerate kinase construct in a bone marrow cell with subcellular resolution. The same instrument was also used to perform the comparative in vitro measurement in dilute buffer and crowded environments. Investigating an ensemble of cells, each cell has its own unique kinetic signature that can differ substantially from the in vitro result. Variations in the cytoplasmic environment are significant modulators of the protein energy landscape. We quantitate these variations with a statistical analysis of multiple cells and compare folding dynamics on the nm length scale with $\mu $m length scale diffusion processes. Cytoplasmic energy landscape modulation may be a candidate for non-genetic regulation of proteins but also challenges protein homeostasis. [Preview Abstract] |
Wednesday, March 23, 2011 1:39PM - 2:15PM |
Q4.00005: Crowding and hydrodynamic interactions likely dominate in vivo macromolecular motion Invited Speaker: To begin to elucidate the principles of intermolecular dynamics in the crowded environment of cells, employing brownian dynamics (BD) simulations, we examined possible mechanism(s) responsible for the great reduction in diffusion constants of macromolecules in vivo from that at infinite dilution. In an Escherichia coli cytoplasm model comprised of 15 different macromolecule types at physiological concentrations, BD simulations of molecular-shaped and equivalent sphere representations were performed with a soft repulsive potential. At cellular concentrations, the calculated diffusion constant of GFP is much larger than experiment, with no significant shape dependence. Next, using the equivalent sphere system, hydrodynamic interactions (HI) were considered. Without adjustable parameters, the in vivo experimental GFP diffusion constant was reproduced. Finally, the effects of nonspecific attractive interactions were examined. The reduction in diffusivity is very sensitive to macromolecular radius with the motion of the largest macromolecules dramatically slowed down; this is not seen if HI dominate. In addition, long-lived clusters involving the largest macromolecules form if attractions dominate, whereas HI give rise to significant, size independent intermolecular dynamic correlations. These qualitative differences provide a testable means of differentiating the importance of HI vs. nonspecific attractive interactions on macromolecular motion in cells. [Preview Abstract] |
Follow Us |
Engage
Become an APS Member |
My APS
Renew Membership |
Information for |
About APSThe American Physical Society (APS) is a non-profit membership organization working to advance the knowledge of physics. |
© 2024 American Physical Society
| All rights reserved | Terms of Use
| Contact Us
Headquarters
1 Physics Ellipse, College Park, MD 20740-3844
(301) 209-3200
Editorial Office
100 Motor Pkwy, Suite 110, Hauppauge, NY 11788
(631) 591-4000
Office of Public Affairs
529 14th St NW, Suite 1050, Washington, D.C. 20045-2001
(202) 662-8700