Protein Folding, Interrupted Kim A. Sharp Science 343, 743 (2014); DOI: 10.1126/science.1249405
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at www.sciencemag.org (this information is current as of February 20, 2014 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/343/6172/743.full.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/343/6172/743.full.html#related This article cites 9 articles, 2 of which can be accessed free: http://www.sciencemag.org/content/343/6172/743.full.html#ref-list-1 This article appears in the following subject collections: Biochemistry http://www.sciencemag.org/cgi/collection/biochem
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on February 20, 2014
This copy is for your personal, non-commercial use only.
PERSPECTIVES projections and recursively determines the rules for the robots to follow. This system is extremely elegant, as it allows the autonomous construction of any predefined structures with simple robots. It is robust to failure because of its decentralization, and is very flexible in its adjustments. The approach by Werfel et al. differs from others in that it develops the lower-level rules from the final structure to be built. Commonly, the reverse approach is pursued in complex systems science, but determining the emergent higher-level result from lower-level rules has proved difficult. Hence, it is also still unclear how termites can construct their impressive castles from the simple behaviors that researchers observe (3, 4).
In both nature’s construction works and the structures created by the robots in the approach of Werfel et al., the properties of the final product are crucial. A termite mound’s architecture can determine the success of a colony (7). Mounds that are better adapted to local environments will, as a rule, have more offspring; thus, improved building rules that are genetically encoded will spread over time through a population. What is different in nature is that it starts with “mutations” in the building rules that are then tested in the evolutionary process. Over the millennia, evolution tested different rules, and what we observe today are those that worked. They might not be perfect, and the algorithms of Werfel et al.
might also show us whether termites could still “learn” from humans. References 1. H. Smeathman, Philos. Trans. R. Soc. London 71, 139 (1781). 2. E. Bonabeau, G. Theraulaz, J. L. Deneubourg, S. Aron, S. Camazine, Trends Ecol. Evol. 12, 188 (1997). 3. S. Camazine et al., Self-Organization in Biological Systems (Princeton Univ. Press, Princeton, NJ, 2001). 4. J. Korb, in Biology of Termites: A Modern Synthesis, D. E. Bignell, Y. Roisin, N. Lo, Eds. (Springer, Dordrecht, Netherlands, 2011), pp. 349–376. 5. J. Werfel, K. Petersen, R. Nagpal, Science 343, 754 (2014). 6. P. P. Grassé, Insectes Soc. 6, 41 (1959). 7. J. Korb, K. E. Linsenmair, Oecologia 118, 183 (1999).
10.1126/science.1250721
BIOCHEMISTRY
An antifreeze protein with a core consisting mostly of ordered water molecules violates widely held ideas about protein stabilization.
Protein Folding, Interrupted Kim A. Sharp
G
lobular proteins start their lives as linear chains of amino acids coming off the ribosome. Proteins must then fold into specific three-dimensional structures to be functional. In 1957, the first such structure, of myoglobin, was determined at atomic resolution (1). Fifty-six years and 90,000-plus protein structures later ( 2), we have a very good idea of the necessary requirements for a stable, specific structure. Key to these requirements is the formation Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. E-mail: [email protected]
of a well-packed, largely anhydrous core (3). Yet, on page 795 of this issue, Sun et al. (4) report an antifreeze protein with a core mostly consisting of water. In globular proteins, the anhydrous protein core provides both structural specificity and energetic stabilization (see the figure, panel A). Burial of apolar amino acid side chains inside the core relieves their unfavorable interaction with water, a process known as the hydrophobic effect (5, 6). Even integral membrane proteins, which function in the nonaqueous lipid bilayer of the membrane and adopt structural motifs that are quite different from those of globular pro-
A
teins, conform to this general principle. Here, the apolar side chains are on the outside of the structure, but by forming close contacts with the apolar lipid tails, they are still removed from water (7). The remarkable structure of the antifreeze protein Maxi reported by Sun et al. flaunts its violation of the anhydrous-core principle. Maxi is a 145 Å–long four-helix bundle formed as a dimer of two-helix monomers. More than 400 highly organized water molecules form an integral part of Maxi’s structure. The water is interleaved as a roughly two-molecule-thick layer between both intra- and intermonomer helix inter-
B
Core questions. In a typical globular protein, the four-helix bundle Rop [Protein Data Bank code: 4DO2 (10)] (A), water (blue) is excluded from the core because of efficient, interdigitated packing of apolar side chains (magenta), surrounded by polar side chains (green). By contrast, highly ordered water remains in the core in the antifreeze protein Maxi reported by
Sun et al. (B). Secondary structure is rendered in orange. Slices through the van der Waals atomic surface taken through the core of the two proteins are shown in wire frame. Van der Waals surface slices produced by Pdb2DSlice and rendered in Pymol (11). In (B), the central third of the 145 Å–long Maxi protein is shown.
www.sciencemag.org SCIENCE VOL 343 14 FEBRUARY 2014 Published by AAAS
743
PERSPECTIVES faces, effectively forming the core of the protein and extending to the ice-binding surface (see the figure, panel B). Sun et al. show that the structure determined from crystallography persists in solution as the active form of the protein. Although ordered water molecules can be detected in most high-resolution x-ray crystallographic structures, they are usually located between replica molecules in the crystal lattice and are probably absent or quite rare when the protein is in native solution conditions, in contrast to the water inside Maxi. That the ordered water structure in Maxi extends to the ice-binding surface is suggestive of the function of this unusual core, given that Maxi, as an antifreeze protein, must bind ice nuclei and inhibit their growth to function. As Sun et al. suggest, this function may have driven the evolution of its unique water core, although clearly such a core is not necessary for antifreeze activity. No other known antifreeze protein has a water core like Maxi’s. When a protein folds, it forms van der Waals (packing) interactions, hydrogen bonds, and electrostatic interactions between charged and polar side chains within the protein. Each of these interactions competes with interactions of comparable strength and number between water and protein in the unfolded state. No such competition exists for the hydrophobic effect:
Entropically favorable release of water upon burying apolar groups unambiguously favors the folded state. Thus, it is widely accepted that stabilization of globular proteins occurs primarily through the hydrophobic effect (8). It is therefore startling that Maxi retains the very structure of water—“semi-clathrate” in the words of Sun et al.—whose formation around apolar groups and subsequent disappearance was deemed to be the hallmark of the hydrophobic effect (5) and pivotal for protein stabilization. Clearly, the balance of interactions that stabilize Maxi is quite different from that used by most proteins. Maxi’s structure has intriguing implications not only for the energetics of protein folding, but for the mechanism and kinetics as well. Examination of the anhydrous core of any protein with its convoluted but well-packed atom-atom interfaces (see the figure, panel A) raises the question of how the water is removed so efficiently during folding. Removal of this water has been proposed as a potential rate-limiting step in protein folding. Two competing mechanisms have been proposed: Either the water is driven out as the protein collapses, or the unfavorable hydration free energy of apolar groups leads to their spontaneous dewetting (9). In the first mechanism, protein packing drives dehydration in the manner of a squeegee, whereas in the second, packing follows dehydration.
Resolution of this question depends on the relative magnitudes of packing and hydration forces, which have proved difficult to determine by experiment or theory. Moreover, proteins may well use both mechanisms. But it seems Maxi did not get the memo on how to fold: It chooses neither route to dehydration. Maxi folds to the point where water not in direct contact with the protein chain is removed from its core. It then arrests further folding to retain a beautifully ordered core of water interleaved between the protein helices. Further analysis of the energetics and kinetics of folding of Maxi will deepen our understanding of protein folding and stabilization. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
J. C. Kendrew et al., Nature 181, 662 (1958). H. M. Berman et al., Nucleic Acids Res. 28, 235 (2000). F. M. Richards, J. Mol. Biol. 82, 1 (1974). T. Sun, F.-H. Lin, R. L. Campbell, J. S. Allingham, P. L. Davies, Science 343, 795 (2014). W. Kauzmann, Adv. Protein Chem. 14, 1 (1959). C. H. Tanford, The Hydrophobic Effect (Wiley, New York, 1973). R. Henderson et al., J. Mol. Biol. 213, 899 (1990). K. A. Dill, Biochem. 29, 7133 (1990). Y. Levy, J. N. Onuchic, Annu. Rev. Biophys. Biomol. Struct. 35, 389 (2006). M. Ambrazi et al., Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 64, 432 (2008). W. L. DeLano, PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, CA, 2002).
10.1126/science.1249405
NEUROSCIENCE
Genetic Resolutions of Brain Convolutions
Genetic analysis of human brain abnormalities aids our understanding of how the cerebral cortex develops and evolves.
Brian G. Rash1 and Pasko Rakic1, 2
C
ortical convolutions—prominent folds on the surface of the human brain—have a long history of speculation (1). The claims range from their function as a bodily cooling system to the attribution of Einstein’s genius to the unusual shape of a single gyrus (the ridge of a cortical fold). Only recently, with advances in molecular genetics and brain imaging techniques, has it become possible to study the development, evolution, and abnormalities of cerebral convolutions in a scientifically 1
Department of Neurobiology, Yale University, New Haven, CT 06510, USA. 2Kavli Institute for Neuroscience, Yale University, New Haven, CT 06510, USA. E-mail pasko.rakic@ yale.edu
744
rigorous manner (2). On page 764 of this issue, Bae et al. (3) show that a specific gene controls the number of gyri that form in a region of the cerebral cortex that includes Broca’s area (the major language area). This begins to pinpoint mechanisms that underlie the development of specialized regions of the human brain and may be relevant to understanding human brain evolution. Bae et al. examined individuals, from three consanguineous families, with abnormal cortical folding restricted to a region surrounding the Sylvian fissure, including Broca’s area within the frontal lobe. Through a genome-wide linkage analysis, the authors traced the abnormality to mutations in the noncoding regulatory region of
the GPR56 gene. GPR56 encodes a protein that functions in cell adhesion and guidance. Mutations caused the peri-Sylvian cortex to be thinner and smoother and exhibit multiple shallow indentations (polymicrogyria). Moreover, the authors discovered a natural, spontaneous mutation in the GPR56 locus, which points to a mechanism that underlies both the formation of cortical maps and the process of gyrification. Bae et al. generated transgenic mice in which different expression patterns of a reporter gene (encoding β-galactosidase) could be driven by part of the noncoding region of GPR56 (a minimal promoter) taken from human, marmoset, dolphin, cat, and mouse. This indicates evolutionary changes in cortical
14 FEBRUARY 2014 VOL 343 SCIENCE www.sciencemag.org Published by AAAS
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at www.sciencemag.org (this information is current as of February 20, 2014 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/343/6172/743.full.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/343/6172/743.full.html#related This article cites 9 articles, 2 of which can be accessed free: http://www.sciencemag.org/content/343/6172/743.full.html#ref-list-1 This article appears in the following subject collections: Biochemistry http://www.sciencemag.org/cgi/collection/biochem
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on February 20, 2014
This copy is for your personal, non-commercial use only.
PERSPECTIVES projections and recursively determines the rules for the robots to follow. This system is extremely elegant, as it allows the autonomous construction of any predefined structures with simple robots. It is robust to failure because of its decentralization, and is very flexible in its adjustments. The approach by Werfel et al. differs from others in that it develops the lower-level rules from the final structure to be built. Commonly, the reverse approach is pursued in complex systems science, but determining the emergent higher-level result from lower-level rules has proved difficult. Hence, it is also still unclear how termites can construct their impressive castles from the simple behaviors that researchers observe (3, 4).
In both nature’s construction works and the structures created by the robots in the approach of Werfel et al., the properties of the final product are crucial. A termite mound’s architecture can determine the success of a colony (7). Mounds that are better adapted to local environments will, as a rule, have more offspring; thus, improved building rules that are genetically encoded will spread over time through a population. What is different in nature is that it starts with “mutations” in the building rules that are then tested in the evolutionary process. Over the millennia, evolution tested different rules, and what we observe today are those that worked. They might not be perfect, and the algorithms of Werfel et al.
might also show us whether termites could still “learn” from humans. References 1. H. Smeathman, Philos. Trans. R. Soc. London 71, 139 (1781). 2. E. Bonabeau, G. Theraulaz, J. L. Deneubourg, S. Aron, S. Camazine, Trends Ecol. Evol. 12, 188 (1997). 3. S. Camazine et al., Self-Organization in Biological Systems (Princeton Univ. Press, Princeton, NJ, 2001). 4. J. Korb, in Biology of Termites: A Modern Synthesis, D. E. Bignell, Y. Roisin, N. Lo, Eds. (Springer, Dordrecht, Netherlands, 2011), pp. 349–376. 5. J. Werfel, K. Petersen, R. Nagpal, Science 343, 754 (2014). 6. P. P. Grassé, Insectes Soc. 6, 41 (1959). 7. J. Korb, K. E. Linsenmair, Oecologia 118, 183 (1999).
10.1126/science.1250721
BIOCHEMISTRY
An antifreeze protein with a core consisting mostly of ordered water molecules violates widely held ideas about protein stabilization.
Protein Folding, Interrupted Kim A. Sharp
G
lobular proteins start their lives as linear chains of amino acids coming off the ribosome. Proteins must then fold into specific three-dimensional structures to be functional. In 1957, the first such structure, of myoglobin, was determined at atomic resolution (1). Fifty-six years and 90,000-plus protein structures later ( 2), we have a very good idea of the necessary requirements for a stable, specific structure. Key to these requirements is the formation Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. E-mail: [email protected]
of a well-packed, largely anhydrous core (3). Yet, on page 795 of this issue, Sun et al. (4) report an antifreeze protein with a core mostly consisting of water. In globular proteins, the anhydrous protein core provides both structural specificity and energetic stabilization (see the figure, panel A). Burial of apolar amino acid side chains inside the core relieves their unfavorable interaction with water, a process known as the hydrophobic effect (5, 6). Even integral membrane proteins, which function in the nonaqueous lipid bilayer of the membrane and adopt structural motifs that are quite different from those of globular pro-
A
teins, conform to this general principle. Here, the apolar side chains are on the outside of the structure, but by forming close contacts with the apolar lipid tails, they are still removed from water (7). The remarkable structure of the antifreeze protein Maxi reported by Sun et al. flaunts its violation of the anhydrous-core principle. Maxi is a 145 Å–long four-helix bundle formed as a dimer of two-helix monomers. More than 400 highly organized water molecules form an integral part of Maxi’s structure. The water is interleaved as a roughly two-molecule-thick layer between both intra- and intermonomer helix inter-
B
Core questions. In a typical globular protein, the four-helix bundle Rop [Protein Data Bank code: 4DO2 (10)] (A), water (blue) is excluded from the core because of efficient, interdigitated packing of apolar side chains (magenta), surrounded by polar side chains (green). By contrast, highly ordered water remains in the core in the antifreeze protein Maxi reported by
Sun et al. (B). Secondary structure is rendered in orange. Slices through the van der Waals atomic surface taken through the core of the two proteins are shown in wire frame. Van der Waals surface slices produced by Pdb2DSlice and rendered in Pymol (11). In (B), the central third of the 145 Å–long Maxi protein is shown.
www.sciencemag.org SCIENCE VOL 343 14 FEBRUARY 2014 Published by AAAS
743
PERSPECTIVES faces, effectively forming the core of the protein and extending to the ice-binding surface (see the figure, panel B). Sun et al. show that the structure determined from crystallography persists in solution as the active form of the protein. Although ordered water molecules can be detected in most high-resolution x-ray crystallographic structures, they are usually located between replica molecules in the crystal lattice and are probably absent or quite rare when the protein is in native solution conditions, in contrast to the water inside Maxi. That the ordered water structure in Maxi extends to the ice-binding surface is suggestive of the function of this unusual core, given that Maxi, as an antifreeze protein, must bind ice nuclei and inhibit their growth to function. As Sun et al. suggest, this function may have driven the evolution of its unique water core, although clearly such a core is not necessary for antifreeze activity. No other known antifreeze protein has a water core like Maxi’s. When a protein folds, it forms van der Waals (packing) interactions, hydrogen bonds, and electrostatic interactions between charged and polar side chains within the protein. Each of these interactions competes with interactions of comparable strength and number between water and protein in the unfolded state. No such competition exists for the hydrophobic effect:
Entropically favorable release of water upon burying apolar groups unambiguously favors the folded state. Thus, it is widely accepted that stabilization of globular proteins occurs primarily through the hydrophobic effect (8). It is therefore startling that Maxi retains the very structure of water—“semi-clathrate” in the words of Sun et al.—whose formation around apolar groups and subsequent disappearance was deemed to be the hallmark of the hydrophobic effect (5) and pivotal for protein stabilization. Clearly, the balance of interactions that stabilize Maxi is quite different from that used by most proteins. Maxi’s structure has intriguing implications not only for the energetics of protein folding, but for the mechanism and kinetics as well. Examination of the anhydrous core of any protein with its convoluted but well-packed atom-atom interfaces (see the figure, panel A) raises the question of how the water is removed so efficiently during folding. Removal of this water has been proposed as a potential rate-limiting step in protein folding. Two competing mechanisms have been proposed: Either the water is driven out as the protein collapses, or the unfavorable hydration free energy of apolar groups leads to their spontaneous dewetting (9). In the first mechanism, protein packing drives dehydration in the manner of a squeegee, whereas in the second, packing follows dehydration.
Resolution of this question depends on the relative magnitudes of packing and hydration forces, which have proved difficult to determine by experiment or theory. Moreover, proteins may well use both mechanisms. But it seems Maxi did not get the memo on how to fold: It chooses neither route to dehydration. Maxi folds to the point where water not in direct contact with the protein chain is removed from its core. It then arrests further folding to retain a beautifully ordered core of water interleaved between the protein helices. Further analysis of the energetics and kinetics of folding of Maxi will deepen our understanding of protein folding and stabilization. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
J. C. Kendrew et al., Nature 181, 662 (1958). H. M. Berman et al., Nucleic Acids Res. 28, 235 (2000). F. M. Richards, J. Mol. Biol. 82, 1 (1974). T. Sun, F.-H. Lin, R. L. Campbell, J. S. Allingham, P. L. Davies, Science 343, 795 (2014). W. Kauzmann, Adv. Protein Chem. 14, 1 (1959). C. H. Tanford, The Hydrophobic Effect (Wiley, New York, 1973). R. Henderson et al., J. Mol. Biol. 213, 899 (1990). K. A. Dill, Biochem. 29, 7133 (1990). Y. Levy, J. N. Onuchic, Annu. Rev. Biophys. Biomol. Struct. 35, 389 (2006). M. Ambrazi et al., Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 64, 432 (2008). W. L. DeLano, PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, CA, 2002).
10.1126/science.1249405
NEUROSCIENCE
Genetic Resolutions of Brain Convolutions
Genetic analysis of human brain abnormalities aids our understanding of how the cerebral cortex develops and evolves.
Brian G. Rash1 and Pasko Rakic1, 2
C
ortical convolutions—prominent folds on the surface of the human brain—have a long history of speculation (1). The claims range from their function as a bodily cooling system to the attribution of Einstein’s genius to the unusual shape of a single gyrus (the ridge of a cortical fold). Only recently, with advances in molecular genetics and brain imaging techniques, has it become possible to study the development, evolution, and abnormalities of cerebral convolutions in a scientifically 1
Department of Neurobiology, Yale University, New Haven, CT 06510, USA. 2Kavli Institute for Neuroscience, Yale University, New Haven, CT 06510, USA. E-mail pasko.rakic@ yale.edu
744
rigorous manner (2). On page 764 of this issue, Bae et al. (3) show that a specific gene controls the number of gyri that form in a region of the cerebral cortex that includes Broca’s area (the major language area). This begins to pinpoint mechanisms that underlie the development of specialized regions of the human brain and may be relevant to understanding human brain evolution. Bae et al. examined individuals, from three consanguineous families, with abnormal cortical folding restricted to a region surrounding the Sylvian fissure, including Broca’s area within the frontal lobe. Through a genome-wide linkage analysis, the authors traced the abnormality to mutations in the noncoding regulatory region of
the GPR56 gene. GPR56 encodes a protein that functions in cell adhesion and guidance. Mutations caused the peri-Sylvian cortex to be thinner and smoother and exhibit multiple shallow indentations (polymicrogyria). Moreover, the authors discovered a natural, spontaneous mutation in the GPR56 locus, which points to a mechanism that underlies both the formation of cortical maps and the process of gyrification. Bae et al. generated transgenic mice in which different expression patterns of a reporter gene (encoding β-galactosidase) could be driven by part of the noncoding region of GPR56 (a minimal promoter) taken from human, marmoset, dolphin, cat, and mouse. This indicates evolutionary changes in cortical
14 FEBRUARY 2014 VOL 343 SCIENCE www.sciencemag.org Published by AAAS
