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The Role of Theory in Advancing 21st-Century Biology: Catalyzing Transformative Research 5 How Do Cells Really Work? Inside each microscopic living cell, thousands of diverse chemical reactions must take place at the right time, in the right places, and in the right order. Scientists can re-create many of these individual reactions, or even a few coupled reactions in the laboratory, but the spacious and uniform conditions of a test tube bear little resemblance to the crowded and highly structured interior of the cell. The sequestration of chemical reactions within cells was probably one of the critical factors in the early evolution of life. Understanding how this complex milieu developed and varies across different life forms will serve as another profound illustration of the ways that evolution has maintained certain common ancestral features throughout life’s history. The theoretical frameworks provided by thermodynamics and the laws of chemical equilibrium have been used productively to study the chemical reactions of life. However, analysis of individual molecules within cells, tissues, and developing embryos reveals important differences from studying these molecules in aqueous solution. To understand the behavior of even familiar macromolecules, biologists need to study them under the conditions found within cells and tissues. These conditions differ from those of typical biochemical experiments in that reactions do not proceed to chemical equilibrium, reaction volumes are small, solutions are crowded and inhomogeneous, the concentrations of enzymes are often higher than that of their substrates, and many reactants are immobilized on membrane or proteinaceous surfaces.
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The Role of Theory in Advancing 21st-Century Biology: Catalyzing Transformative Research UNDERSTANDING THE CELL Cells and organisms are crowded and complicated (Figure 5-1). Although the macromolecular structures within cells must be self-assembling, they perform this self-assembly following elaborate temporal expression and spatial localization of their individual constituents. The highly energy-requiring events of a living cell require the synthesis of large macromolecules and their specific localization against concentration gradients and through crowded solutions. The flux of energy that moves through the system allows the reactions to be held away from equilibrium, and this is an essential characteristic of living systems. Understanding these processes requires an appreciation of nonequilibrium thermodynamics, a situation that can be sustained when energy is constantly added, as it is during life. In chemistry one of the most powerful, unifying concepts is equilibrium thermodynamics, the principle that allows a prediction with confidence that ice left at room temperature will melt and that water put in a freezer will become ice. Under any given set of conditions, a chemical system will tend to change its properties, including temperature, pressure, and concentration of reactive chemical species, toward a particular stable state called “equilibrium.” If the system is perturbed slightly away from its equilibrium state, it will robustly return to equilibrium. If the system is left alone, it will remain at equilibrium indefinitely. There are excellent, accurate mathematical formalisms for calculating and predicting equilibrium states of even FIGURE 5-1 Artist’s rendition of the crowded conditions within a cell. Illustration by David S. Goodsell, Scripps Research Institute.
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The Role of Theory in Advancing 21st-Century Biology: Catalyzing Transformative Research very complex physical and chemical systems, using a fairly small number of “state variables.” Most of biologists’ understanding of the biochemical reactions in living cells has come from experiments with purified enzymes and substrates studied in isolation. For the most part, these experiments have been done under conditions that are not mimicked within living cells and interpreted using assumptions that are not appropriate for living cells. For example, an isolated, purified enzyme placed in a test tube with its substrate will catalyze the conversion of the substrate into product until equilibrium is reached. However, the actual behavior of the enzyme and its substrate inside a living cell is, in most cases, different for several reasons, including, for example, that the products of reactions are usually then further transformed. Living cells do not operate at chemical equilibrium. A cell or organism at equilibrium would be dead. Additional assumptions that are appropriate for reactions in dilute aqueous solutions but not in cells and tissues are: that reaction volumes are infinite; that the solutions in which reactions occur are dilute, well defined, and homogenous; that molecules collide due to diffusional motion; and that concentrations of substrates are higher than concentrations of enzymes. The inappropriateness of these simplifying assumptions for all living systems is discussed in this chapter and underlines the need for new approaches to better understand the biochemistry of the living cell. A DIFFERENT VIEW OF CELL CHEMISTRY In cells, reaction volumes are finite. Cell volumes vary—for example, a mycobacterium is about 10−11 microliters and a Xenopus laevis egg is about 0.5 microliters. In the bacterium Escherichia coli, a protein present at 1 nanomolar concentration, a concentration at which many enzymes are studied in the laboratory, is calculated to be present at 0.6 copies per cell. The volume within a 50-nanometer vesicle such as the vesicles involved in protein transport within cells is 6 × 10–20 liters, which means that one free proton within such a small vesicle would yield a pH of 6.0, and 50 free protons would yield a pH of 5.0. Therefore, only a few events would be required to acidify a vesicle involved in endocytosis, for example. Furthermore, cells vary greatly in shape, from roughly spherical to elongated. Cells are highly organized and many are specialized for specific functions. In some cases structure obviously correlates with function, such as in the polarization of epithelial cells.
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The Role of Theory in Advancing 21st-Century Biology: Catalyzing Transformative Research In cells, solutions are not dilute, well defined, or homogenous. Instead, the interior of a cell is 17 to 35 percent protein by weight, and the individual proteins are likely to be associated with each other and with other molecules in complicated ways. The effects of this macromolecular crowding are extensive. Mobility of molecules, including water, is decreased, with larger molecules more affected than smaller molecules. “Nonspecific” interactions between proteins—such as those interactions that do not normally occur in aqueous solutions—are enhanced, and “specific” interactions occur more readily. For example, since chemical equilibrium theory is based on activities, not concentrations, the equilibrium constant for dimerization of a 40,000-kilodalton molecular weight protein is 10 to 40 times greater in a cell than in dilute solution, and its tetramerization is 1,000- to 100,000-fold greater. The ability of cellular machinery to localize individual proteins and other macromolecules within the cell in specific ways will lend apparent specificity to otherwise nonspecific reactions. For example, there are many different pairs of “SNARE” proteins (Figure 5-2) that are known to facilitate fusion between intracellular membrane compartments. Within cells the many different membrane fusion events that are facilitated are highly specific, with endosomal membranes fusing with lysosomal membranes and not, for example, with mitochondrial or FIGURE 5-2 Much of the specificity of SNARE-SNARE fusion is likely to derive from specific cellular localization. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology 7:631-643. SNAREs—Engines for Membrane Fusion, R. Jahn and R. H. Scheller, copyright 2006.
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The Role of Theory in Advancing 21st-Century Biology: Catalyzing Transformative Research nuclear membranes. However, purified SNARE proteins mediate fusion reactions relatively promiscuously, showing affinities for each other that do not correlate with their known partnerships within the cell. A conceptual framework based on results in aqueous solution would lead to a search for additional specificity “factors”; in this case, however, it appears that the specificity most likely resides in the intricate mechanisms of the localization and orientation of these disparate SNARE proteins within the cell. Therefore, development of conceptual frameworks that take into account the crowded interior of the cell and guide experimentation to determine how organization and localization are achieved has great potential. Within cells, diffusional motion is highly restricted. Movement of groups of individual molecules can be measured inside cells by photobleaching fluorescent molecules in a limited area and monitoring the rate at which they exchange with unbleached molecules. These and other measurements have revealed that proteins move 10 to 50 times more slowly inside the cell than in aqueous solution, with many proteins displaying a completely immobile subpopulation. These restrictions to movement are strongly size dependent, with larger complexes being almost immobile. What limits the diffusion of these molecules? Is it nonspecific interactions with the high concentration of other proteins, or specific interactions that cause many proteins to function in much higher-order complexes than previously suspected, or sieving through the network of the cytoskeleton, or reduced water activity due to macromolecular crowding? To the extent known so far, all these factors come into play. Poor diffusion of molecules within cells necessitates that any specific intracellular localization must be accomplished by specific transport of the mRNAs that encode the proteins, the proteins themselves, or both. In cells, concentrations of enzymes are often higher than their substrates. The concentrations of many steady-state metabolites are lower than the measured binding constants for the enzymes that process them, predicting that there should be little free substrate. How, then, are multistep reactions accomplished? “Substrate channeling” is a common solution (Figure 5-3). From carbamoyl phosphate synthetase to transfer ribonucleic acid (tRNA) synthetases, enzymes that catalyze individual steps of multistep reactions have been found to be co-localized or present in large complexes. Such complexes might, in fact, exclude nonchanneled substrates. In the case of tRNA synthetases, the direct introduction of free tRNA into cells does not result in its incorporation into charged tRNA synthetase, even though such reactions occur readily among purified components in aqueous solution. The reality of the inside of a living cell, which has poor diffusion, immobilized reacting groups, and high degrees of localization, changes the
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The Role of Theory in Advancing 21st-Century Biology: Catalyzing Transformative Research FIGURE 5-3 Substrate channeling. Upper image: Cartoon depicting substrate channeling in tryptophan synthase. Reprinted from Trends in Biochemical Sciences, Vol. 17, J. Ovadi and P. A. Srere, Channel Your Energies, Page 3, Copyright 1992, with permission from Elsevier. Lower image: Structure of the tryptophan synthase complex, which the substrate tunnel highlighted. SOURCE: The Molecular Basis of Substrate Channeling in Journal of Biological Chemistry, Vol. 274, by E. W. Miles, S. Rhee, and D. R. Davies. Copyright 1999. Reproduced with permission of American Society for Biochemistry and Molecular Biology via Copyright Clearance Center.
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The Role of Theory in Advancing 21st-Century Biology: Catalyzing Transformative Research outcomes of interactions between molecules. Take, for example, the well-studied example of an RNA polymerase transcribing a messenger RNA from a double-stranded DNA template. Even in solution, it is unlikely that a polymerase molecule, tracking along the template DNA strand, actually follows a helical path around the DNA molecule. In complex situations—for example, when a newly synthesized strand of RNA becomes associated with ribosomes, or when there are trailing peptide chains, or when dealing with spliceosomes and their complex machinery—it is highly unlikely that these dangling structures would twist around the DNA as the polymerase follows such a helical path. Instead, the DNA template is pulled through a relatively immobile polymerase, removing its helicity as it goes. Therefore, positive supercoiling (an overabundance of helical turns) accumulates behind the transcription complex and negative supercoiling in front of the complex. Conceptually, these turns could be easily removed, especially in a linear DNA template, by diffusional forces that allowed the DNA to spin on its long axis, much as one can unwind an overtwisted telephone cord by allowing the handset to dangle. Nevertheless, within cells, even for linear DNA molecules such as the 40,000 base pair T7 DNA phage genome, transient positive and negative supercoiling occur concomitantly with transcription. In living cells, enzymes termed “topoisomerases” are required to solve these problems during transcription. What prevents the diffusional release of DNA underwinding and overwinding within a cell? Two possibilities are the association of the nominally free DNA ends with subcellular structures or macromolecules that bind along DNA in a manner that is independent of the DNA sequence. Are restrictions to diffusion within the cell so severe that even the spinning of DNA molecules along their long axes is limited? In addition to the complexity of the environment and reaction pathways of the molecules in a cell, we know that individual reactions are embedded in networks of reactions. The “metabolic network” of a cell is a term now used to describe all of these activities and interactions. It is remarkable, in the light of the foregoing discussion of the complexities of the chemistry of the cell, that the reactions can be intermeshed so beautifully, using substrate channeling as well as other yet unknown mechanisms. Enzymes and other proteins are often localized within cells, either via specific association with a membrane-bound organelle such as the mitochondrion, endoplasmic reticulum, or the membranous vesicles shown by freeze-etch electron microscopy in Figure 5-4 or via other less understood processes such as association with proteinaceous assemblages such as “P bodies” or “nuclear speckles.” For human hepatocytes, the intracellular area presented by internal membranes is approximately 50 times the area of the plasma membrane. The cytoskeleton, even when undecorated with auxiliary proteins, is expected to present another set of surfaces greater than that of the plasma membrane. What are the consequences of the
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The Role of Theory in Advancing 21st-Century Biology: Catalyzing Transformative Research FIGURE 5-4 (A) Surface structure of a COPII vesicle, involved in secretory traffic from the endoplasmic reticulum to the Golgi apparatus, compared to the clathrin coat of a vesicle involved in endocytosis. Deep-etch platinum shadowed electron microscopic images are shown. (B) Images can be used to reconstruct the iterative molecular structures that form their surface coats. Permission granted by Randy Schekman. Surface Structure of the COPII-coated Vesicle. Proceedings of the National Academy of Sciences, USA 98:13705-13709. K. Matsuoka, R. Schekman, L. Orci, and J. E. Heuser. 2001.
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The Role of Theory in Advancing 21st-Century Biology: Catalyzing Transformative Research localization of cellular substituents on the surface of such structures for enzymatic activity and specificity? Are the assemblages found within cells merely storage forms of the enzymes of interest, as is often speculated? Or is it possible that ordered arrays of reagents, in the nuclear matrix, on the surface of membranous vesicles or on the exposed surfaces of proteinaceous arrays provide the advantages of surface catalysis to biological systems? In mature and developing organisms, local interactions among cells are mediated by complex local context. For example, in a developing Drosophila embryo, extremely high local concentrations of morphogens are formed and shape cell differentiation and mobility. Developing neurons will establish synaptic connections in response to subtle gradients. Understanding these cues requires not only identifying all the molecules involved but also developing analytical interpretive theories for their roles and testing those theories with, for example, high-definition and quantitative visualization techniques. CONCLUSION Understanding the activities and specificities of molecules and larger arrays within cells and tissues will require additional techniques from biophysics, microscopy, materials science, microfluidics, and computational biology. A particular need is the development of microscopy that bridges the gap between fluorescent light microscopy and electron microscopy. In addition to technological advances, the use of simulations of the movements of individual molecules (for example, using the Monte Carlo approach) and the development of theories that incorporate the nonequilibrium conditions of the cell could fuel new scientific advances.