We combine experiment and computer simulation to show how macromolecular crowding dramatically affects the structure,function, and folding landscape of phosphoglycerate kinase (PGK). Fluorescence labeling shows that compact states of yeast PGK are populated as the amount of crowding agents (Ficoll 70) increases. Coarse-grained molecular simulations reveal three compact ensembles: C (crystal structure), CC (collapsed crystal), and Sph (spherical compact). With an adjustment for viscosity, crowded wild-type PGK and fluorescent PGK are about 15 times or more active in 200 mg/ml Ficoll than in aqueous solution. Our results suggest a previously undescribed solution to the classic problem of how the ADP and diphosphoglycerate binding sites of PGK come together to make ATP: Rather than undergoing a hinge motion, the ADP and substrate sites are already located in proximity under crowded conditions that mimic the in vivo conditions under which the enzyme actually operates. We also examine T-jump unfolding of PGK as a function of crowding experimentally. We uncover a nonmonotonic folding relaxation time vs. Ficoll concentration. Theory and modeling explain why an optimum concentration exists for fastest folding. Below the optimum, folding slows down because the unfolded state is stabilized relative to the transition state. Above the optimum, folding slows down because of increased viscosity.
A. Dhar, A. Samiotakis, S. Ebbinghaus, L. Nienhaus, D. Homouz, M. Gruebele and M. S. Cheung, " Structure, function and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding", PNAS, 107, 17586-17591 (2010).
Featured in the Cover Story "Tight Quarters" in Chemical & Engineering News, Nov 29th, 2010 issue. [link]
In vitro biochemical reactions are most often studied in dilute solution, a poor mimic of the intracellular space of eukaryotic cells, which are crowded with mobile and immobile macromolecules. Such crowded conditions exert volume exclusion and other entropic forces that have the potential to impact chemical equilibria and reaction rates. In this article, we use characterized and ubiquitous molecule calmodulin (CaM) and a combination of theoretical and experimental approaches to address how crowding impacts CaM's conformational plasticity. CaM is a dumbbell-shaped molecule that contains four EF hands (two in the N-lobe and two in the C-lobe) that each could bind Ca2+, leading to stabilization of certain substates that favor interactions with other target proteins. Using coarse-grained molecular simulations, we explored the distribution of CaM conformations in the presence of crowding agents. These predictions, in which crowding effects enhance the population of compact structures, were then confirmed in experimental measurements using fluorescence resonance energy transfer techniques of donor- and acceptor-labeled CaM under normal and crowded conditions. Using protein reconstruction methods, we further explored the folding-energy landscape and examined the structural characteristics of CaM at free-energy basins. We discovered that crowding stabilizes several different compact conformations, which reflects the inherent plasticity in CaM's structure. From these results, we suggest that the EF hands in the C-lobe are flexible and can be thought of as a switch, while those in the N-lobe are stiff, analogous to a rheostat. New combinatorial signaling properties may arise from the product of the differential plasticity of the two distinct lobes of CaM in the presence of crowding. We discuss the implications of these results for modulating CaM's ability to bind Ca2+ and target proteins.
D. Homouz, H. Sanabria, M. N. Waxham, M. S. Cheung, “Modulation of calmodulin plasticity by the effect of macromolecular crowding”, J. Mol. Biol. 391, 933-943 (2009).
How the crowded environment inside cells affects the structures of proteins with aspherical shapes is a vital question because many proteins and protein-protein complexes in vivo adopt anisotropic shapes. Here we address this question by combining computational and experimental studies of a football-shaped protein (i.e., Borrelia burgdorferi VlsE) in crowded, cell-like conditions. The results show that macromolecular crowding affects protein-folding dynamics as well as overall protein shape. In crowded milieus, distinct conformational changes in VlsE are accompanied by secondary structure alterations that lead to exposure of a hidden antigenic region (green segment in Figure right). 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.
Dirar Homouz, Michael Perham, Antonios Samiotakis, Margaret S. Cheung, and Pernilla Wittung-Stafshede, "Crowded, cell-like environment induces shape changes in aspherical protein ", Proc. Natl. Acad. Sci. U. S. A., 105, 11754-11759, (2008).
It was featured in Chemical and Engineering News and in Nature as one of the Research Highlights of 2008.
To investigate the consequences of macromolecular crowding on the behavior of a globular protein, we performed a combined experimental and computational study on the 148-residue, single-domain protein, Desulfovibrio desulfuricans apo-flavodoxin. In vitro thermal unfolding experiments, as well as assessment of native and denatured structures, were probed using far-UV circular dichroism (CD) in the presence of various amounts of Ficoll 70, an inert spherical crowding agent. Ficoll 70 has a concentration-dependent effect on the thermal stability of apo-flavodoxin. As judged by CD, addition of Ficoll 70 causes an increase in the amount of secondary structure in the native-state ensemble but only minor effects on the denatured state. Theoretical calculations, based on an off-lattice model for an apoflavodoxin protein and hard-sphere particles for Ficoll 70, are in good agreement with the in vitro data. The simulations demonstrate that, in the presence of 25 % volume occupancy of spheres, native flavodoxin is thermally stabilized and the free energy landscape shifts to favor more compact structures in both native and denatured states. It is revealed that the native-state compaction originates in stronger interactions between the helices and the central beta-sheet, as well as by less fraying in the terminal helices. This is the first study to demonstrate that macromolecular crowding has structural effects on the folded ensemble of polypeptides.
L. Stagg, S.-Q.Zhang, M. S. Cheung, P. Wittung-Stafshede, "Molecular crowding enhances native structure and stability of alpha/beta; protein flavodoxin", Proc. Natl. Acad. Sci. U. S. A., 104, 18976-18981, (2007).
Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents φc as well as of crowding agent geometry (sphere or spherocylinder) at high φc. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. Our test-tube experiments confirmed that apoflavodoxin's time-resolved folding path is modulated by crowding agent geometry. Macromolecular crowding effects may be a tool for the manipulation of protein-folding and function in living cells.
D. Homouz, L. Stagg, P. Wittung-Stafshede, M. S. Cheung,“Macromolecular crowding modulates folding mechanism of α/β protein apoflavodoxin”, Biophys. J., 96, 671-680 (2009).
Flavodoxin adopts the common repeat β/α topology and folds in a complex kinetic reaction with intermediates. To better understand this reaction, we analyzed a set of Desulfovibrio desulfuricans apoflavodoxin variants with point mutations in most secondary structure elements by in vitro and in silico methods. By equilibrium unfolding experiments, we first revealed how different secondary structure elements contribute to overall protein resistance to heat and urea. Next, using stopped-flow mixing coupled with far-UV circular dichroism, we probed how individual residues affect the amount of structure formed in the experimentally detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central β-strand 3 (i.e., at β-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in folding nuclei growth correlates with the in vitro burst-phase circular dichroism amplitude. In addition, φ-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the β-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central β-sheet directs initial misfolding, while proper alignment on both sides of β-strand 3 is necessary for productive folding.
L. Stagg, A. Samiotakis, M. S. Cheung, P. Wittung-Stafshede, “Residue specific analysis of frustration in folding landscape of repeat β/α protein apoflavodoxin”, J. Mol. Biol, 396, 75-89 (2010)
We use all-atomistic molecular dynamics simulations to study hydrophobic interactions of hexane in nanosized water droplets where the hydrogen bonding network of water molecules is disrupted at the surface. As a result of the competition between the energetics of a hexane molecule and the distribution of water molecules that lost the ability to form hydrogen bonds at the boundary, all-trans-hexane molecules are statistically favored at the surface of a nanosized water droplet and such a statistical trend increases as the size of a nano water droplet decreases. Changes in the radial distribution and the orientation of water molecules surrounding hexane in nanosized water droplets over bulk water are indicative of the finite-size effects on the structural distribution of a short, topologically complex hydrocarbon chain.
D. Homouz, B. Hoffman, M. S. Cheung, “Hydrophobic interactions of hexane in nanosized water droplets”, J. Phys. Chem. B, 113, 12337-12342 (2009).
The behavior of biopolymers in nano-sized confinement is investigated using coarse-grained models and molecular simulations. We address the effects of geometry of a confinement and the wall-protein interactions on protein folding dynamics. By measuring folding rates and dissecting structural properties of the transition states in nano-sized spheres and ellipsoids, we are able to justify the best form of a confinement in which the rates of folding kinetics are most enhanced. This knowledge in identifying optimal conditions for reactions will have a broad impact in nanotechnology and pharmaceutical sciences.
S.-Q.Zhang and M. S. Cheung. "Manipulating Biopolymer Dynamics by Anisotropic Nanoconfinement", Nano Letters 7, 3438-3442(2007).
It was featured in Research Highlights by Nature Nanotechnology 2007.
The flexibility in the structure of calmodulin (CaM) allows its binding to over 300 target proteins in the cell. To investigate the structure-function relationship of CaM, we combined methods of computer simulation and experiments based on circular dichroism (CD) to investigate the structural characteristics of CaM that influence its target recognition in crowded cell-like conditions. We developed a unique multiscale solution of charges computed from quantum chemistry, together with protein reconstruction, coarse-grained molecular simulations, and statistical physics, to represent the charge distribution in the transition from apoCaM to holoCaM upon calcium binding. Computationally, we found that increased levels of macromolecular crowding, in addition to calcium binding and ionic strength typical of that found inside cells, can impact the conformation, helicity and the EF hand orientation of CaM. Because EF hand orientation impacts the affinity of calcium binding and the specificity of CaM's target selection, our results may provide unique insight into understanding the promiscuous behavior of calmodulin in target selection inside cells.
Q. Wang, K.-C. Liang, A. Czader, M. N. Waxham and M. S. Cheung, "The Effect of Macromolecular Crowding, Ionic Strength and Calcium Binding on Calmodulin Dynamics", PLoS Comput Biol 7(7): e1002114, (2011)
One of the projects is to characterize protein structures in the presence of chemical denaturants, e.g., urea. Determining this structure is considered a difficult problem in molecular biology. We have successfully resolved this problem by developing a new computational method, MultiSCAAL, that cleverly integrates both low-resolution and high resolution protein models. The Trp-cage protein structure from our MultiSCAAL simulations is in better agreement with the NOE distances reported from NMR experiments over the structure from all-atomistic replica exchange molecular dynamics (AA-REMD). MultiSCAAL can effectively capture the changes in side chain orientations of Trp-cage that can flip out of the hydrophobic pocket in the presence of urea and water molecules (Figure below), which AA-REMD cannot. In this regard, MultiSCAAL is a promising and effective sampling scheme for investigating the types of chemical interference which present a great challenge when modeling protein interactions in a living cell.
(Left) A representative structure of Trp–cage under 8M urea of the most dominant cluster from MultiSCAAL simulations where a coarse-grained protein model (shaded) is used to facilitate all-atomistic molecular dynamics simulations. Water molecules located in the hydrophobic core are shown in van der Waals representation. (Right) A representative structure of Trp–cage under the same urea condition of the most dominant cluster from the standard AA-REMD. Residues I4 and W6 are shown in yellow, while protons HE3 in W6 and HG2 in I4 are colored silver their distances are indicated
A. Samiotakis, D. Homouz, M. S. Cheung, "Multiscale Investigation of Chemical Interference in Proteins". J. Chem. Phys.132, 175101 (2010).