8.The dissociation constant is on the order of micromolar (Nissim, A., Hoogenboom, H. R., Tomlinson, I. M., Flynn, G., Midgley, C., Lane, D., and Winter, G. 1994. Antibody fragments from a “single pot” phage display library as immunochemical reagents. EMBO J. 13:692–98).
9.Dissociation constant on the order of nanomolar (Griffiths, A. D., Williams, S. C., Hartley, O., Tomlinson, I. M., Waterhouse, P., Crosby, W. L., Kontermann, R. E., Jones, P. T., Low, N. M., Allison, T. J., and Winter, G. 1994. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 13:3245–60).
10.Other proteins besides antibodies have been used to demonstrate the same point. For example, rather than antibodies, a protein called knottin was used by Winter to generate a shape-space library. About five hundred million binding sites had to be screened to find one that stuck to a test protein with modest affinity (Smith, G. P., Patel, S. U., Windass, J. D., Thornton, J. M., Winter, G., and Griffiths, A. D. 1998. Small binding proteins selected from a combinatorial repertoire of knottins displayed on phage. J. Mol. Biol. 277:317–32). A roughly similar result was seen when part of the sequence of a small bacterial protein was randomized to generate a library containing about forty million members, and when a library of fifty million artificial “minibodies” was probed for binding to a protein called interleukin (Nord, K., Gunneriusson, E., Uhlen, M., and Nygren, P. A. 2000. Ligands selected from combinatorial libraries of protein A for use in affinity capture of apolipoprotein A-1M and taq DNA polymerase. J. Biotechnol. 80:45–54; Martin, F., Toniatti, C., Salvati, A. L., Venturini, S., Ciliberto, G., Cortese, R., and Sollazzo, M. 1994. The affinity-selection of a minibody polypeptide inhibitor of human interleukin-6. EMBO J. 13:5303–9).
11.Most proteins are present in the cell at well below millimolar concentrations, so in order for two proteins to spend the majority of their time bound to each other, micromolar dissociation constants would be required to form even a “weak, transient” complex (Nooren, I. M., and Thornton, J. M. 2003. Structural characterisation and functional significance of transient protein-protein interactions. J. Mol. Biol. 325:991–1018). Dissociation constants on the order of micromolar seem to be required to detect interactions in yeast two-hybrid assays (Golemis, E. A., and Serebriiskii, I. 1996. Identification of protein-protein interactions. In Coligan, J. E., ed. Current protocols in protein science. Brooklyn, N.Y.: John Wiley & Sons, Inc; Estojak, J., Brent, R., and Golemis, E. A. 1995. Correlation of two-hybrid affinity data with in vitro measurements. Mol. Cell Biol. 15:5820–29).
12.As discussed in Chapter 7, there are different kinds of mutations—deletions, duplications, and so on. But point mutation represents the conceptually simplest, most straightforward route. This calculation uses consensus values for important variables. One could certainly imagine other scenarios for making a new protein-binding site, for example by first invoking gene duplication and then point mutation. But those are either unlikely to help much (Behe, M. J., and Snoke, D. W. 2004. Simulating evolution by gene duplication of protein features that require multiple amino acid residues. Protein Sci. 13:2651–64) or likely to involve special circumstances that amount to a Just-So story. All alternative scenarios would have to confront the fact that no new binding sites have turned up in the best-studied evolutionary cases of malaria and HIV, as described later in the text.
13.Even though protein-binding sites often involve a score of amino acids on each of the partners, experiments have shown that only a fraction of those are important for having the two proteins stick to each other. (For example, see Braden, B. C., and Poljak, R. J. 1995. Structural features of the reactions between antibodies and protein antigens. FASEB J. 9:9–16; Lo Conte, L., Chothia, C., and Janin, J. 1999. The atomic structure of protein-protein recognition sites. J. Mol. Biol. 285:2177–98; Ma, B., Elkayam, T., Wolfson, H., and Nussinov, R. 2003. Protein-protein interactions: structurally conserved residues distinguish between binding sites and exposed protein surfaces. Proc. Natl. Acad. Sci. USA 100:5772–77.) In terms of the swimming pool analogy, the five or six residues represent bumps and magnets that are aligned very nicely; if enough are aligned, then it doesn’t matter so much if other features aren’t aligned, as long as they don’t actively block the surfaces from coming together.
14.Axe, D. D. 2004. Estimating the prevalence of protein sequences adopting functional enzyme folds. J. Mol. Biol. 341:1295–1315.
15.Geretti, A. M. 2006. HIV-1 subtypes: epidemiology and significance for HIV management. Curr. Opin. Infect. Dis. 19:1–7. Rodrigo, A. G. 1999. HIV evolutionary genetics. Proc. Natl. Acad. Sci. USA 96:10559–61. Total body burden of the number of copies of HIV RNA is estimated to be much higher, about 1011 (Haase, A. T., Henry, K., Zupancic, M., Sedgewick, G., Faust, R. A., Melroe, H., Cavert, W., Gebhard, K., Staskus, K., Zhang, Z. O., Dailey, P. J., Balfour, H. H., Jr., Erice, A., and Perelson, A. S. 1996. Quantitative image analysis of HIV-1 infection in lymphoid tissue. Science 274:985–89). The effective population size is estimated at 500 to 105 (Althaus, C. L., and Bonhoeffer, S. 2005. Stochastic interplay between mutation and recombination during the acquisition of drug resistance mutations in human immunodeficiency virus type 1. J. Virol. 79:13572–78).
16.Rodrigo, A. G., Shpaer, E. G., Delwart, E. L., Iversen, A. K., Gallo, M. V., Brojatsch, J., Hirsch, M. S., Walker, B. D., and Mullins, J. I. 1999. Coalescent estimates of HIV-1 generation time in vivo. Proc. Natl. Acad. Sci. USA 96:2187–91.
17.Coffin, J. M. 1995. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science 267:483–89.
18.For example, see Viard, M., Parolini, I., Rawat, S. S., Fecchi, K., Sargiacomo, M., Puri, A., and Blumenthal, R. 2004. The role of glycosphingolipids in HIV signaling, entry and pathogenesis. Glycocon. J. 20:213–22; Bandivdekar, A. H., Velhal, S. M., and Raghavan, V. P. 2003. Identification of CD4-independent HIV receptors on spermatozoa. Am. J. Reprod. Immunol. 50:322–27. The virus also is taken up by dendritic cells, whose job is to transport them to lymphoid organs. For a short review, see Stebbing, J., Gazzard, B., and Douek, D. C. 2004. Where does HIV live? N. Engl. J. Med. 350:1872–80.
19.HIV, Human Immunodeficiency Virus, is thought to have originated from a similar virus infecting other primates, SIV, Simian Immunodeficiency Virus. Is the ability of the virus to change host species a major biochemical novelty? No. Apparently several changes in a small, variable region of one SIV protein were sufficient to allow SIV from sooty mangabeys to successfully infect Rhesus macaques (Demma, L. J., Logsdon, J. M., Vanderford, T. H., Feinberg, M. B., and Staprans, S. I. 2005. SIVsm quasispecies adaptation to a new simian host. PLoS Pathol. 1:e3).
20.What if something useful did happen, but so far has escaped detection? That of course can never be ruled out, but is unlikely. By definition, if something “useful” to the virus occurred, the mutant virus would increase rapidly in the population. Since the progression of HIV in the world is monitored closely, such an event would likely be noticed.
21.Wang (Wang, J. 2002. Protein recognition by cell surface receptors: physiological receptors versus virus interactions. Trends Biochem. Sci. 27:122–26) argues that viruses must bind their receptors more strongly than the normal physiological receptor ligand. However, he points out that normal ligands don’t bind very strongly on an individual basis, relying on the avidity of multivalent binding to compensate. So a viral protein that bound a cell surface protein with a modest dissociation constant of one micromolar or better should be sufficient. Micromolar dissociation constants would be expected to be found in a shape-space library of 108 surfaces. That many mutant viruses would be present in a single individual each day. However, the mutations would not likely be clustered in a coherent patch, as they are in the shape-space libraries that Winter developed.
22.Sarafianos, S. G., Das, K., Hughes, S. H., and Arnold, E. 2004. Taking aim at a moving target: designing drugs to inhibit drug-resistant HIV-1 reverse transcriptases. Curr. Opin. Struct. Biol. 14:716–30.
23.Tie, Y., Boross, P. I., Wang, Y. F., Gaddis, L., Liu, F., Chen, X., Tozser, J., Harrison, R. W., and Weber, I. T. 2005. Molecular basis for substrate recognition and drug resistance from 1.1 to 1.6 angstroms resolution crystal structures of HIV-1 protease mutants with substrate analogs. FEBS J. 272:5265–77.
24.The evasion of the immune system by HIV is also biochemically simple, involving point mutations in the outer proteins of the virus coat, which apparently causes antibodies that had been produced against the virus to bind less strongly. For example, see Frost, S. D., Wrin, T., Smith, D. M., Pond, S. L., Liu, Y., Paxinos, E., Chappey, C., Galovich, J., Beauchaine, J., Petropoulos, C. J., Little, S. J., and Richman, D. D. 2005. Neutralizing antibody responses drive the evolution of human immunodeficiency virus type 1 envelope during recent HIV infection. Proc. Natl. Acad. Sci. USA 102:18514–19.
25.Lenski, R. E. 2004. Phenotypic and genomic evolution during a 20,000-generation experiment with the bacterium Escherichia coli. Plant Breeding Reviews 24:225–65; Cooper, T. F., Rozen, D. E., and Lenski, R. E.2003. Parallel changes in gene expression after 20,000 generations of evolution in Escherichia coli. Proc. Natl. Acad. Sci. USA 100:1072–77; Cooper, V. S., Schneider, D., Blot, M., and Lenski, R. E. 2001. Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B. J. Bacteriol. 183:2834–41; Vulic, M., Lenski, R. E., and Radman, M. 1999. Mutation, recombination, and incipient speciation of bacteria in the laboratory. Proc. Natl. Acad. Sci. USA 96:7348–51.
26.“Experimental populations of Escherichia coli have evolved for 20,000 generations in a uniform environment. Their rate of improvement, as measured in competitions with the ancestor in that environment, has declined substantially over this period…. Instead, the pronounced deceleration in its rate of fitness improvement indicates that the population early on incorporated most of those mutations that provided the greatest gains, and subsequently relied on beneficial mutations that were fewer in number, smaller in effect, or both” (de Visser, J. A., and Lenski, R. E. 2002. Long-term experimental evolution in Escherichia coli. XI. Rejection of non-transitive interactions as cause of declining rate of adaptation. BMC Evol. Biol. 2:19).
8 Objections to the Edge
1.Proteins that interact with chemicals, especially small molecules in metabolism, also constitute a separate category. One reason for placing these in a separate category is that metabolites are often present at much higher concentrations in the cell than are proteins.
2.For example, some viral proteins stick to cell proteins that normally trigger defensive action, either by alerting the immune system that the cell has been invaded or by tripping a mechanism that makes compromised cells self-destruct (Tortorella, D., Gewurz, B. E., Furman, M. H., Schust, D. J., and Ploegh, H. L. 2000. Viral subversion of the immune system. Annu. Rev. Immunol. 18:861–926; Gale, M., Jr., and Foy, E. M. 2005. Evasion of intracellular host defence by hepatitis C virus. Nature 436:939–45).
3.Deoxy HbS has a solubility of about 3 millimolar (Behe, M. J., and Englander, S. W. 1979. Mixed gelation theory. Kinetics, equilibrium and gel incorporation in sickle hemoglobin mixtures. J. Mol. Biol. 133:137–60). It’s dissociation constant, however, is much higher due to excluded volume effects (Ivanova, M., Jasuja, R., Kwong, S., Briehl, R. W., and Ferrone, F. A.2000. Nonideality and the nucleation of sickle hemoglobin. Biophys. J. 79:1016–22).
4.Rollins, C. T., Rivera, V. M., Woolfson, D. N., Keenan, T., Hatada, M., Adams, S. E., Andrade, L. J., Yaeger, D., van Schravendijk, M. R., Holt, D. A., Gilman, M., and Clackson, T. 2000. A ligand-reversible dimerization system for controlling protein-protein interactions. Proc. Natl. Acad. Sci. USA 97:7096–7101.
5.Stronger binding isn’t necessarily better for a protein. One protein called CA that coats the RNA genome of HIV has amino acids that decrease the binding of the protein to itself, apparently allowing it to do its job better (del Alamo, M., Neira, J. L., and Mateu, M. G. 2003. Thermodynamic dissection of a low affinity protein-protein interface involved in human immunodeficiency virus assembly. J. Biol. Chem. 278:27923–29).
6.Proteins sometimes take on the altered shapes of other proteins in neurological diseases such as Alzheimer’s (Dobson, C., M. 2002. Protein-misfolding diseases: getting out of shape. Nature 418:729–30). Also, some proteins that bind copies of themselves are thought to have arisen by “domain swapping.” That is, areas called “domains” that had been in contact in a single protein instead bind to the analogous region of a second copy of the protein. This scenario does not propose that new binding sites arose, just that pre-existing binding sites got mixed up (Rousseau, F., Schymkowitz, J. W., and Itzhaki, L., S. 2003. The unfolding story of three-dimensional domain swapping. Structure 11:243–51).
7.Actually for many organisms the mutation rate per cell duplication is even lower, more like one in 1010. However, for animals like humans, several hundred generations of cells pass between the birth of a parent and the birth of a child, so the rate of inherited nucleotide mutations per generation is about one in 108 (Drake, J. W., Charlesworth, B., Charlesworth, D., and Crow, J. F.1998. Rates of spontaneous mutation. Genetics
8.This assumes a genome of 108 nucleotides or larger.
9.Voet, D., and Voet, J. G. 2004. Biochemistry. New York: J. Wiley & Sons, p.14.
10.Kauffman, S. A. 1995. At home in the universe: the search for laws of self-organization and complexity. New York: Oxford University Press. Camazine(2001) gives many persuasive examples of self-organizing behavior in biological systems (almost entirely at the organismal, rather than molecular, level) but gives no examples of the self-organization of evolution (Camazine, S., et al. 2001. Self-organization in biological systems. Princeton, N.J.: Princeton University Press).
11.Shapiro, J. A., and Sternberg R. V. 2005. Why repetitive DNA is essential to genome function. Biological Reviews 80:227–50.
12.Some scientists such as Shapiro restrict the term “Darwinian” to mutations of small effect, and thus think of big events such as gene duplications or transpositions as “non-Darwinian.”
13.Han, J. S., Szak, S. T., and Boeke, J. D. 2004. Transcriptional disruption by the L1 retrotransposon and implications for mammalian transcriptomes. Nature 429:268–74.
14.Fondon, J. W., III, and Garner, H. R. 2004. Molecular origins of rapid and continuous morphological evolution. Proc. Natl. Acad. Sci. USA 101:18058–63.
15.Ronshaugen, M., McGinnis, N., and McGinnis, W. 2002. Hox protein mutation and macroevolution of the insect body plan. Nature 415:914–17.
16.Schneider, D., Duperchy, E., Coursange, E., Lenski, R. E., and Blot, M.2000. Long-term experimental evolution in Escherichia coli. IX. Characterization of insertion sequence-mediated mutations and rearrangements. Genetics 156:477–88.
17.Maxwell, J. C. 1952. The scientific papers of James Clerk Maxwell. New York: Dover Publications; “Ether,” Encyclopaedia Brittanica, ninth edition, pp. 763–75.
18.One objection to the shipwrecked-sailor analogy might be the following: We know humans are intelligent agents, so we can conclude that a sailor might have arranged some features of the island, but we can’t conclude design if we don’t have a human designer in the wings. To understand why that reasoning is wrong, instead of a deserted island, just change the example to a tellurian spaceship that crashes on an unexplored planet. When the marooned astronaut spots a wrecked, alien ship over a hill, the same reasoning applies, now with a nonhuman intelligent agent.
19.“The purposeful or inventive arrangement of parts or details,” www.thefreedictionary.com/design. In Darwin’s Black Box (p. 193), I defined design as “the purposeful arrangement of parts.”
20.For example, suppose the shipwrecked sailor arranged rocks throughout the island to point to various constellations in the night sky. You might easily not notice the arrangement, even if you saw the individual rocks.
9 The Cathedral and the Spandrels
1.Kirschner, M., and Gerhart, J. 2005. The plausibility of life: resolving Darwin’s d
ilemma. New Haven, Conn.: Yale University Press, p. 53.
2.Laughon, A., and Scott, M. P. 1984. Sequence of a Drosophila segmentation gene: protein structure homology with DNA-binding proteins. Nature 310:25–31. Subsequent work has shown that homeodomain helix-turn-helix motifs are somewhat different from bacterial ones (Branden, C., and Tooze, J.1999. Introduction to protein structure, 2nd ed. New York: Garland Publishing /Taylor and Francis).
3.Davidson, E. H. 2000. Genomic regulatory systems: development and evolution. San Diego: Academic Press, pp. 11–12.
4.A good introductory description of Drosophila development can usually be found in any recent textbook of molecular biology or developmental biology. For example, Alberts, B. 2002. Molecular biology of the cell, 4th ed. New York: Garland Science; Gilbert, S. F., Singer, S. R., Tyler, M. S., and Kozlowski,R. N. 2003. Developmental biology, 7th ed. Sunderland, Mass.: Sinauer Associates.
5.The messenger RNA for the proteins is deposited.
6.Gehring, W. J. 1996. The master control gene for morphogenesis and evolution of the eye. Genes Cells 1:11–15.
7.Wagner, G. P., and Schlosser, G. 2004. Modularity in development and evolution. Chicago: University of Chicago Press; Callebaut, W., and Rasskin-Gutman, D. 2005. Modularity: understanding the development and evolution of natural complex systems. Cambridge, Mass.: MIT Press.
8.Callebaut, W., and Rasskin-Gutman, D. 2005, sections III and IV.
9.There are four different kinds of nucleotides in DNA (A, C, G, and T). So the probability of matching a six-nucleotide-long binding site is one in four to the sixth power, which is one in 4,096.
10.Stone, J. R., Wray, G. A. 2001. Rapid evolution of cis-regulatory sequences via local point mutations. Mol. Biol. Evol. 18:1764–70.
11.A further study confirmed the notion that regulatory protein switch sequences are a dime a dozen: Almost one in a hundred of all random eighteenth-nucleotide-long DNA fragments had the ability to increase the rate at which a test gene was expressed. (Edelman, G. M., Meech, R., Owens, G. C., and Jones, F. S. 2000. Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity. Proc. Natl. Acad. Sci. USA 97:3038–43).
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