High Concentrations Of Macromolecules Research Articles

Conformational Sampling of Peptides in Cellular Environments

Biological systems provide a complex environment that can be understood in terms of its dielectric properties. High concentrations of macromolecules and cosolvents effectively reduce the dielectric constant of cellular environments, thereby affecting the conformational sampling of biomolecules. To examine this effect in more detail, the conformational preference of alanine dipeptide, poly-alanine, and melittin in different dielectric environments is studied with computer simulations based on recently developed generalized Born methodology. Results from these simulations suggest that extended conformations are favored over α-helical conformations at the dipeptide level at and below dielectric constants of 5–10. Furthermore, lower-dielectric environments begin to significantly stabilize helical structures in poly-alanine at ɛ=20. In the more complex peptide melittin, different dielectric environments shift the equilibrium between two main conformations: a nearly fully extended helix that is most stable in low dielectrics and a compact, V-shaped conformation consisting of two helices that is preferred in higher dielectric environments. An additional conformation is only found to be significantly populated at intermediate dielectric constants. Good agreement with previous studies of different peptides in specific, less-polar solvent environments, suggest that helix stabilization and shifts in conformational preferences in such environments are primarily due to a reduced dielectric environment rather than specific molecular details. The findings presented here make predictions of how peptide sampling may be altered in dense cellular environments with reduced dielectric response.

Effect of molecular crowding on DNA polymerase activity

Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo; however, the molecular conditions within live cells are inherently crowded. The present study investigates the effect of molecular crowding on DNA polymerase activity using polyethylene glycol PEG of various molecular weights as a crowding agent. Polymerase activity assays under various conditions demonstrated that the activities of T7 and Taq DNA polymerases depend on the molecular weight and concentration of the crowding agent. Furthermore, equilibrium and kinetic analyses demonstrated that the binding affinity and catalytic activity of the polymerase increase and decrease, respectively, with increasing PEG concentrations. Based on quantitative parameters of the polymerase reactions, we improved the efficiency of PCR amplification under conditions of molecular crowding. These results suggest that quantitative measurements of biomolecular structure and function are useful for understanding the behavior of biomolecules in vivo and for biotechnology applications in vitro.

Separating the Contribution of Translational and Rotational Diffusion to Protein Association

The association of two proteins is preceded by a mutual diffusional search in solution. The role of translational and rotational diffusion in this process has been studied theoretically for many years. However, systematic experimental verification of theoretical results is still lacking. We report here measurements of association rates of the proteins beta-lactamase (TEM) and beta-lactamase inhibitor protein (BLIP) in solutions of glycerol and poly(ethylene glycol) of increasing viscosity. We also measured translational and rotational diffusion in the same solutions, using fluorescence correlation spectroscopy and fluorescence anisotropy, respectively. It is found that in glycerol both translational and rotational diffusion rates are inversely dependent on viscosity, as predicted by the classical Stokes-Einstein relations, while the association rate depends nonlinearly on viscosity. In contrast, the association rate depends only weakly on the viscosity of the polymer solutions, which results in a similar weak dependence of k(on) on viscosity. The data are modeled using the theory of diffusion-limited association. Deviations from the theory are explained by a short-range solute-induced repulsion between the proteins in glycerol solution and an attractive depletion interaction generated by the polymers. These results open the way to the creation of a unified framework for all nonspecific effects involved in the protein association process, as well as to better theoretical understanding of these effects. Further, they reflect on the complex factors controlling protein association within the crowded environment of cells and suggest that a high concentration of macromolecules does not significantly impede protein association.

Contribution of molecular chaperones to protein folding in the cytoplasm of prokaryotic and eukaryotic cells.

While it is clear that many unfolded proteins can attain their native state spontaneously in vitro, the efficiency of such folding is usually limited to conditions far removed from those encountered within cells. Two properties of the cellular environment are expected to enhance strongly the propensity of incompletely folded polypeptides to misfold and aggregate: the crowding effect caused by the high concentration of macromolecules, and the close proximity of nascent polypeptide chains emerging from polyribosomes. However, in the living cell, non-productive protein folding is in many, if not most, cases prevented by the action of a highly conserved set of proteins termed molecular chaperones. In the cytoplasm, the Hsp70 (heat-shock protein of 70 kDa) and chaperonin families of molecular chaperones appear to be the major contributors to efficient protein folding during both normal conditions and adverse conditions such as heat stress. Hsp70 chaperones recognize and shield short, hydrophobic peptide segments in the context of non-native polypeptides and probably promote folding by decreasing the concentration of aggregation-prone intermediates. In contrast, the chaperonins interact with and globally enclose collapsed folding intermediates in a central cavity where efficient folding can proceed in a protected environment. For a number of proteins, folding requires the co-ordinated action of both of these molecular chaperones.

Macromolecular crowding and confinement in cells exposed to hypertonicity

The nonideal properties of solutions containing high concentrations of macromolecules can result in enormous increases in the activity of the individual macromolecules. It has been proposed that molecular crowding and confinement occur in cells and are major determinants of the activity of the proteins and other intracellular macromolecules. This concept has important implications for cell volume regulation because, under crowded conditions, relatively small changes in concentration, consequent to alterations of water content, lead to large changes in macromolecular activity. This review considers several aspects of macromolecular crowding and confinement, including: 1) the physical chemical principles involved; 2) in vitro demonstrations of the effects; 3) relation to water activity; 4) estimates of the actual intracellular activity of water and macromolecules; 5) relation to osmotic regulation in various types of cells, including bacteria, red blood cells, and complex nucleated cells; and 6) the relation to inorganic ions and organic osmolytes in cells stressed by hypertonicity. We conclude that, while there is compelling evidence for important effects of molecular crowding in vitro and in red blood cells, the role of macromolecular crowding and confinement in osmotic regulation of more complex cells is an open question that deserves the extensive attention it is currently receiving.

Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli

The very high concentration of macromolecules within cells can potentially have an overwhelming effect on the thermodynamic activity of cellular components because of excluded volume effects. To estimate the magnitudes of such effects, we have made an experimental study of the cytoplasm of Escherichia coli. Parameters from cells and cell extracts are used to calculate approximate activity coefficients for cytoplasmic conditions. These calculations require a representation of the sizes, concentrations and effective specific volumes of the macromolecules in the extracts. Macromolecule size representations are obtained either by applying a two-phase distribution assay to define a related homogeneous solution or by using the molecular mass distribution of macromolecules from gel filtration. Macromolecule concentrations in cytoplasm are obtained from analyses of extracts by applying a correction for the dilution that occurs during extraction. That factor is determined from experiments based upon the known impermeability of the cytoplasmic volume to sucrose in intact E. coli. Macromolecule concentrations in the cytoplasm of E. coli in either exponential or stationary growth phase are estimated to be ≈0.3 to 0.4 g/ml. Macromolecule specific volumes are inferred from the composition of close-packed precipitates induced by polyethylene glycol. Several well-characterized proteins which bind to DNA ( lac repressor, RNA polymerase) are extremely sensitive to changes in salt concentration in studies in vitro, but are insensitive in studies in vivo. Application of the activity coefficients from the present work indicates that at least part of this discrepancy arises from the difference in excluded volumes in these studies. Applications of the activity coefficients to solubility or to association reactions are also discussed, as are changes associated with cell growth phase and osmotic or other effects. The use of solutions of purified macromolecules that emulate the crowding conditions inferred for cytoplasm is discussed.

Activity of cyclic-AMP phosphodiesterase in permeabilised cells of Bakers' yeast.

Yeast cyclic-AMP phosphodiesterases were assayed in situ, using cells permeabilised with cytochrome c, to get information about the kinetics of these enzymes at the high concentrations of macromolecules occurring in vivo. Protamine treatment was not suitable, because it perturbed the intracellular localisations of both the (Mg-dependent) low-Km enzyme and the (EDTA-insensitive) high-Km enzyme. The pH-dependence of Km and V for EDTA-insensitive activity in situ agreed well with the behaviour of pure high-Km enzyme, except that near pH 8 Hofstee plots were bent slightly upwards both for activity in situ and with crude broken-cell preparations. Hofstee plots of Mg-dependent activity in situ were distinctly concave, and could be resolved mathematically into two activities, one (accounting for about 30% of the Mg-dependent V) with a Km close to the value in vitro of 0.2 microM, and the other with an apparent Km of 3 microM. The 3 microM Km activity probably represents a fraction of the low-Km enzyme that is particle-bound at the high protein concentrations occurring in situ.

ONTOGENY OF ESTRADIOL-BINDING SITES IN RAT BRAIN. II. CHARACTERISTICS OF A NEONATAL BINDING MACROMOLECULE

Fetal and neonatal rat brain cytosols contain high concentrations of macromolecules which stereospecifically bind tritiated estradiol in vitro. Certain physical-chemical properties of this perinatal binding system were characterized and compared with properties of the soluble cytoplasmic estradiol-binding protein present in adult female hypothalamus and uterus. The estimated total estradiol-binding capacity of the neonatal brain cytosol, although saturable, is relatively high: 4000 pmoles estradiol per g cytosol protein, approximately 200-fold greater than the binding capacity of adult hypothalamic cytosol previously reported by Eisenfeld. Some other features of the neonatal binding substance which differ from those of the adult binding protein include: 1) migration as an approximately “4S” peak in a 5–20% sucrose density gradient; 2) failure to bind diethylstilbesterol; 3) absence of regional concentration within the brain; 4) failure to bind in vitro to rat DNA in the presence of 0.14M NaCl. While diffe...

Macromolecules May Inhibit Diffusion of Hemoglobin from Lysing Erythrocytes by Exclusion of Solvent

An interpretation is provided for the phenomenon of macromolecule inhibition of hemoglobin release from hemolyzing erythrocytes. High concentrations of macromolecules, which are extensively hydrated, remove solvent volume accessible to the diffusing hemoglobin, thus retarding the diffusion. This explanation considerably alters previous interpretations of the mechanism of immune lysis of cells.