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Biopolymers
599
Biopolymers Chemical and biological approaches for understanding form and function Editorial overview Anne Dell*, Michael Famulok† and Barbara Imperiali‡ Addresses *Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK; e-mail: [email protected] † Department of Chemistry, 18-287, Kekulé Inst. für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany; e-mail: [email protected] ‡ Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; e-mail: [email protected] Current Opinion in Chemical Biology 2000, 4:599–601 1367-5931/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations GPI glycosylphosphatidylinositol HSPG heparan sulfate proteoglycan
In this section of Current Opinion in Chemical Biology, an emphasis on interdisciplinary approaches towards understanding the form and function of ever more complex biological systems prevails. The complementary application of both chemical and biochemical synthetic methods now provides access to a diverse array of biopolymers. These materials can serve as the substrates for in-depth analyses of the relationships between structure and function using state-of-the-art spectroscopic tools and biological assays. In addition, the exploitation of biologically inspired materials for novel, non-native functions such as in biosensor applications is highlighted. Another theme in this issue concerns the macromolecular interactions that govern the vast majority of biological processes. Research aimed at understanding how biopolymers behave as social molecules that are responsive to each other’s presence lies at the heart of chemical biology. Two reviews in this section, one on heparan sulfate proteoglycans, the other on bacterial pili address this issue. Nucleic acid biopolymers are covered in the first two reviews. The first review focuses on DNA replication and approaches in chemical biology to probe the function of DNA polymerases. The second article introduces the concept of using DNA as a material in nanotechnolgy. Reliable transfer of genetic information is essential for the survival of all living species. DNA polymerases play the central role in the DNA replication machinery and because of this the polymerases have to be highly specific enzymes making less than one error in a million nucleotide additions. DNA polymerases have been studied extensively and several research groups have recently taken up the challenge of designing novel nucleotide analogs to investigate the function of these complex enzymes. The review
by Kool (pp 602–608) highlights recent advances in this area and provides an intriguing summary of new insights into DNA polymerase function gained by the application of tailor-made nucleotide analogs. Early research on DNA polymerases was mainly influenced by the perception that the complementarity of Watson–Crick hydrogen bonds was the primary determinant for the ability of DNA polymerases to synthesize DNA with high fidelity. Initial attempts to probe this paradigm were devoted to altering the sequence of hydrogen donor and acceptors in newly designed nucleobases. These investigations led to the development of new base pairs and an extension of the genetic alphabet. Kool also describes the rational design of new isosteric nucleobase shape analogs. Although these analogs cannot form hydrogen bonds they maintain a geometry similar to their natural counterparts and are replicated by DNA polymerases with remarkable efficiency and selectivity. These findings have now led to a model in which geometric selection serves as a principal determinant of DNA replication fidelity. Biological processes require precisely tuned molecular interactions and highly organized modes of communication. The ability of biopolymers and other molecules to self-assemble forms the basis of any ‘living entity’. Recently, researchers have begun to take advantage of the principles of biopolymeric self-assembly for the construction a variety of nanometer-sized functional devices. The review by Niemeyer (pp 609–618) discusses recent developments in the growing field of nanostructured supramolecular DNA-scaffolds. Amongst all the biopolymers, DNA is probably the most eminently suited for this purpose; DNA can be easily functionalized through chemical methods and it can serve as the basis for obtaining supramolecular hybrid molecules in which individual DNA-strands are linked to other molecules such as proteins, organic or inorganic complexes, and semiconductor nanoclusters. In this way, proteins and other biopolymers, spatially assembled on surfaces or microarrays, become accessible. These defined sets of biopolymers are important for the genome-wide determination of function in high-throughput formats. Research in DNA-based biomolecular nanotechnology also explores the coupling of biological with electronic systems, material science, and the development of new biosensors. In the next three reviews, the focus turns to biopolymers conjugated with complex carbohydrates. In light of the growing recognition of the importance of glycoconjugates
600
Biopolymers
in numerous aspects of chemical biology, a critical research objective in this area includes understanding how to exploit both chemical and biochemical synthetic approaches for the preparation of homogeneous samples of material for biological and biophysical studies. The review by Flitsch (pp 619–625) addresses the current challenges in this chemically demanding area by assessing the current state of glycopolymer synthesis. The development of methods for oligosaccharide synthesis continues to represent a major challenge because the unique saccharide monomer units and the desired glycosidic linkages cannot be assembled through a common chemical approach in the same way that polypeptides and oligonucleotides can be prepared. Advances in the availability of powerful chemical coupling methods and reactivity tuning, together with the development of strategies that render the chemistry amenable to execution on a solid phase, as well as biosynthetic approaches that employ glycosyltransferases and glycosidases finally start to provide hope that there is light at the end of the oligosaccharide synthesis tunnel! Recent successes with the assembly of complex glycolipids, proteoglycans and glycoproteins are also illustrated in this review and two of these glycoconjugates form the focus of the next two articles by Sasisekharan and by Kinoshita. Heparan sulfate proteoglycans (HSPGs) constitute one of the most widespread and versatile families of eukaryotic biopolymers found on the surfaces of cells and in the extracellular matrices. The HS component of HSPGs is known to bind to a remarkable range of extracellular proteins including growth factors and their receptors, proteases and their inhibitors, cytokines, chemokines and collectins. Heparan sulfate polysaccharides are linear structures comprising tandem repeats of a disaccharide unit containing a uronic acid and an acetamido sugar. Structural and functional diversity is afforded by either inversion at a single carbon in some of the uronic acid residues, substitution of the acetyl group of the acetamido sugar with sulfate or addition of sulfate to selected hydroxyl groups. Establishing exactly where these modifications have occurred in the polymer is a daunting task but is an essential prerequisite to understanding the roles of HSPGs in biological systems. Until recently, HSPGs were studied in relatively few specialist laboratories. Now, however, they are becoming more mainstream, thanks partly to the dedicated efforts of those who have persevered against heavy odds with their isolation and characterization and partly because of increasing evidence that HSPGs play a vital role in modulating and regulating receptor–ligand interactions in, for example, growth factor mediated processes. The review by Sasisekharan and Venkataraman (pp 626–631) highlights recent work on HSPGs, focusing in particular on technological developments that are facilitating understanding of the molecular interactions occurring in HSPG–ligand–receptor macromolecular assemblies. Many eukaryotic proteins, particular those in protozoa, are anchored into the outer leaflet of the cytoplasmic membrane
by glycosylphosphatidylinositol (GPI) anchors. The review by Kinoshita and Inoue (pp 632–638) focuses on the rapid progress that is being made in unraveling the biosynthesis of these highly complex glycolipids and highlights possible enzyme targets whose specific inhibition might provide the basis for novel chemotherapy against diseases such as trypanosomiasis. In keeping with the theme of macromolecular assemblies, this review includes recent work in which the enzyme complex catalyzing the first step in GPI biosynthesis, the generation of GlcNAc–PI from UDP–GlcNAc and PI, has been isolated and characterized. Remarkably this GPI–GlcNAc transferase consists of at least six proteins, some of which are probably involved in regulating the glycosyltransferase activity. Elucidating the mechanism and control of this key step in GPI anchor synthesis is clearly a challenge for future work at the interface of chemistry and biology. An important frontier in chemical biology concerns the interactions of biopolymers with the supramolecular architecture of the lipid bilayer. Whereas the article by Kinoshita investigates the bilayer localizing GPI glycoconjugate, the review by Bechinger (pp 639–644) presents the current experimental approaches for understanding the interactions of hydrophobic and amphipathic polypeptides with biological membranes. The study of protein–membrane interactions from the foundation of relatively simple systems is essential because it provides the conceptual framework for predicting and understanding the behavior of far larger and more complex membrane-associated proteins. This specific goal is particularly critical in light of the fact that approaching 30% of the open reading frames code for membrane-associated proteins, yet, there are relatively few high-resolution structural analyses of these targets because of difficulties with protein expression and crystallization. Dougherty (pp 645–652) presents a valuable tool for probing structure/function relationships in integral membrane proteins such as ion channels and neuroreceptors. The nonsense suppression methodology for incorporating unnatural amino acids into proteins is a powerful technique that harnesses the multidisciplinary skills of biological chemists. This technique allows the incorporation of a wide variety of unnatural amino acids at virtually any position in a protein primary sequence. The advantage of the method over conventional site-directed mutagenesis is that specifically tailored amino acids, for example those with finely tuned electronic or steric properties, can be incorporated to ask very precise questions about the function of a particular residue in a complex biopolymer structure. The method was originally conceptualized for the in vitro translation of novel proteins. However, the in vivo adaptation of the methodology that functions in intact Xenopus oocytes is particularly well suited for the study of ion channels and neuroreceptors because modern electrophysiology techniques in whole-cell assays allow for functional measurements to be made on as little as 10 attomoles of protein. Therefore, while the nonsense
Editorial overview Dell, Famulok and Imperiali
suppression approach is often hampered by protein production levels, in the current context, ample amounts of protein can be produced to carry out detailed analyses of the role of specific amino acids in specific membranebound protein families. Gram-negative bacteria have to adhere to carbohydrate ligands on the surface of host cells. This is achieved via a lectin domain at the distal end of rod-like surface appendages called pili. The final review, by Knight, Berglund and Choudhury (pp 653–660), provides a fascinating insight into early events in the pili assembly pathway. Pili rods contain thousands of copies of pilus subunits, assembled into a giant hollow cylinder. The pilus subunits are synthesized in the cytoplasm and understanding how they make their way to the bacterial surface in an ordered fashion, without premature aggregation, has been a major challenge in microbiology. In particular, the involvement of periplasmic chaperones in the assembly pathway has been well established. The chaperones prevent mis-aggregation and precipitation of newly synthesized pilus subunits by plucking them from the cytoplasmic membrane, assisting their correct folding and escorting them to the outer membrane where ‘usher’
601
pore-forming proteins provide a platform for assembly and export. The first two crystal structures of chaperone–pilus complexes have now been reported and have proved to be enormously rewarding. Knight and colleagues explain in their review how the concepts of donor-strand complementation and donor-strand exchange, which are suggested by the molecular architecture, can help to explain both chaperone function and pilus biogenesis. In summary, the reviews in this section once again reveal the reward of interdisciplinary research in chemical biology. The merging of synthetic organic chemistry with biology expands the toolbox for the investigation of biopolymer structure and function in molecular detail, thereby facilitating our understanding of complex biological processes. One of the major goals of the approaches highlighted here is using synthetic or semi-synthetic components as molecular flashlights to gain insight into cellular processes that cannot be fully studied with the existing methodologies of structural biology, cell biology and biochemistry. The technological advances derived from this knowledge will increasingly enable the application of biopolymers for practical purposes both in vivo and ex vivo.
Biopolymers Chemical and biological approaches for understanding form and function Editorial overview Anne Dell*, Michael Famulok† and Barbara Imperiali‡ Addresses *Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK; e-mail: [email protected] † Department of Chemistry, 18-287, Kekulé Inst. für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany; e-mail: [email protected] ‡ Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; e-mail: [email protected] Current Opinion in Chemical Biology 2000, 4:599–601 1367-5931/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations GPI glycosylphosphatidylinositol HSPG heparan sulfate proteoglycan
In this section of Current Opinion in Chemical Biology, an emphasis on interdisciplinary approaches towards understanding the form and function of ever more complex biological systems prevails. The complementary application of both chemical and biochemical synthetic methods now provides access to a diverse array of biopolymers. These materials can serve as the substrates for in-depth analyses of the relationships between structure and function using state-of-the-art spectroscopic tools and biological assays. In addition, the exploitation of biologically inspired materials for novel, non-native functions such as in biosensor applications is highlighted. Another theme in this issue concerns the macromolecular interactions that govern the vast majority of biological processes. Research aimed at understanding how biopolymers behave as social molecules that are responsive to each other’s presence lies at the heart of chemical biology. Two reviews in this section, one on heparan sulfate proteoglycans, the other on bacterial pili address this issue. Nucleic acid biopolymers are covered in the first two reviews. The first review focuses on DNA replication and approaches in chemical biology to probe the function of DNA polymerases. The second article introduces the concept of using DNA as a material in nanotechnolgy. Reliable transfer of genetic information is essential for the survival of all living species. DNA polymerases play the central role in the DNA replication machinery and because of this the polymerases have to be highly specific enzymes making less than one error in a million nucleotide additions. DNA polymerases have been studied extensively and several research groups have recently taken up the challenge of designing novel nucleotide analogs to investigate the function of these complex enzymes. The review
by Kool (pp 602–608) highlights recent advances in this area and provides an intriguing summary of new insights into DNA polymerase function gained by the application of tailor-made nucleotide analogs. Early research on DNA polymerases was mainly influenced by the perception that the complementarity of Watson–Crick hydrogen bonds was the primary determinant for the ability of DNA polymerases to synthesize DNA with high fidelity. Initial attempts to probe this paradigm were devoted to altering the sequence of hydrogen donor and acceptors in newly designed nucleobases. These investigations led to the development of new base pairs and an extension of the genetic alphabet. Kool also describes the rational design of new isosteric nucleobase shape analogs. Although these analogs cannot form hydrogen bonds they maintain a geometry similar to their natural counterparts and are replicated by DNA polymerases with remarkable efficiency and selectivity. These findings have now led to a model in which geometric selection serves as a principal determinant of DNA replication fidelity. Biological processes require precisely tuned molecular interactions and highly organized modes of communication. The ability of biopolymers and other molecules to self-assemble forms the basis of any ‘living entity’. Recently, researchers have begun to take advantage of the principles of biopolymeric self-assembly for the construction a variety of nanometer-sized functional devices. The review by Niemeyer (pp 609–618) discusses recent developments in the growing field of nanostructured supramolecular DNA-scaffolds. Amongst all the biopolymers, DNA is probably the most eminently suited for this purpose; DNA can be easily functionalized through chemical methods and it can serve as the basis for obtaining supramolecular hybrid molecules in which individual DNA-strands are linked to other molecules such as proteins, organic or inorganic complexes, and semiconductor nanoclusters. In this way, proteins and other biopolymers, spatially assembled on surfaces or microarrays, become accessible. These defined sets of biopolymers are important for the genome-wide determination of function in high-throughput formats. Research in DNA-based biomolecular nanotechnology also explores the coupling of biological with electronic systems, material science, and the development of new biosensors. In the next three reviews, the focus turns to biopolymers conjugated with complex carbohydrates. In light of the growing recognition of the importance of glycoconjugates
600
Biopolymers
in numerous aspects of chemical biology, a critical research objective in this area includes understanding how to exploit both chemical and biochemical synthetic approaches for the preparation of homogeneous samples of material for biological and biophysical studies. The review by Flitsch (pp 619–625) addresses the current challenges in this chemically demanding area by assessing the current state of glycopolymer synthesis. The development of methods for oligosaccharide synthesis continues to represent a major challenge because the unique saccharide monomer units and the desired glycosidic linkages cannot be assembled through a common chemical approach in the same way that polypeptides and oligonucleotides can be prepared. Advances in the availability of powerful chemical coupling methods and reactivity tuning, together with the development of strategies that render the chemistry amenable to execution on a solid phase, as well as biosynthetic approaches that employ glycosyltransferases and glycosidases finally start to provide hope that there is light at the end of the oligosaccharide synthesis tunnel! Recent successes with the assembly of complex glycolipids, proteoglycans and glycoproteins are also illustrated in this review and two of these glycoconjugates form the focus of the next two articles by Sasisekharan and by Kinoshita. Heparan sulfate proteoglycans (HSPGs) constitute one of the most widespread and versatile families of eukaryotic biopolymers found on the surfaces of cells and in the extracellular matrices. The HS component of HSPGs is known to bind to a remarkable range of extracellular proteins including growth factors and their receptors, proteases and their inhibitors, cytokines, chemokines and collectins. Heparan sulfate polysaccharides are linear structures comprising tandem repeats of a disaccharide unit containing a uronic acid and an acetamido sugar. Structural and functional diversity is afforded by either inversion at a single carbon in some of the uronic acid residues, substitution of the acetyl group of the acetamido sugar with sulfate or addition of sulfate to selected hydroxyl groups. Establishing exactly where these modifications have occurred in the polymer is a daunting task but is an essential prerequisite to understanding the roles of HSPGs in biological systems. Until recently, HSPGs were studied in relatively few specialist laboratories. Now, however, they are becoming more mainstream, thanks partly to the dedicated efforts of those who have persevered against heavy odds with their isolation and characterization and partly because of increasing evidence that HSPGs play a vital role in modulating and regulating receptor–ligand interactions in, for example, growth factor mediated processes. The review by Sasisekharan and Venkataraman (pp 626–631) highlights recent work on HSPGs, focusing in particular on technological developments that are facilitating understanding of the molecular interactions occurring in HSPG–ligand–receptor macromolecular assemblies. Many eukaryotic proteins, particular those in protozoa, are anchored into the outer leaflet of the cytoplasmic membrane
by glycosylphosphatidylinositol (GPI) anchors. The review by Kinoshita and Inoue (pp 632–638) focuses on the rapid progress that is being made in unraveling the biosynthesis of these highly complex glycolipids and highlights possible enzyme targets whose specific inhibition might provide the basis for novel chemotherapy against diseases such as trypanosomiasis. In keeping with the theme of macromolecular assemblies, this review includes recent work in which the enzyme complex catalyzing the first step in GPI biosynthesis, the generation of GlcNAc–PI from UDP–GlcNAc and PI, has been isolated and characterized. Remarkably this GPI–GlcNAc transferase consists of at least six proteins, some of which are probably involved in regulating the glycosyltransferase activity. Elucidating the mechanism and control of this key step in GPI anchor synthesis is clearly a challenge for future work at the interface of chemistry and biology. An important frontier in chemical biology concerns the interactions of biopolymers with the supramolecular architecture of the lipid bilayer. Whereas the article by Kinoshita investigates the bilayer localizing GPI glycoconjugate, the review by Bechinger (pp 639–644) presents the current experimental approaches for understanding the interactions of hydrophobic and amphipathic polypeptides with biological membranes. The study of protein–membrane interactions from the foundation of relatively simple systems is essential because it provides the conceptual framework for predicting and understanding the behavior of far larger and more complex membrane-associated proteins. This specific goal is particularly critical in light of the fact that approaching 30% of the open reading frames code for membrane-associated proteins, yet, there are relatively few high-resolution structural analyses of these targets because of difficulties with protein expression and crystallization. Dougherty (pp 645–652) presents a valuable tool for probing structure/function relationships in integral membrane proteins such as ion channels and neuroreceptors. The nonsense suppression methodology for incorporating unnatural amino acids into proteins is a powerful technique that harnesses the multidisciplinary skills of biological chemists. This technique allows the incorporation of a wide variety of unnatural amino acids at virtually any position in a protein primary sequence. The advantage of the method over conventional site-directed mutagenesis is that specifically tailored amino acids, for example those with finely tuned electronic or steric properties, can be incorporated to ask very precise questions about the function of a particular residue in a complex biopolymer structure. The method was originally conceptualized for the in vitro translation of novel proteins. However, the in vivo adaptation of the methodology that functions in intact Xenopus oocytes is particularly well suited for the study of ion channels and neuroreceptors because modern electrophysiology techniques in whole-cell assays allow for functional measurements to be made on as little as 10 attomoles of protein. Therefore, while the nonsense
Editorial overview Dell, Famulok and Imperiali
suppression approach is often hampered by protein production levels, in the current context, ample amounts of protein can be produced to carry out detailed analyses of the role of specific amino acids in specific membranebound protein families. Gram-negative bacteria have to adhere to carbohydrate ligands on the surface of host cells. This is achieved via a lectin domain at the distal end of rod-like surface appendages called pili. The final review, by Knight, Berglund and Choudhury (pp 653–660), provides a fascinating insight into early events in the pili assembly pathway. Pili rods contain thousands of copies of pilus subunits, assembled into a giant hollow cylinder. The pilus subunits are synthesized in the cytoplasm and understanding how they make their way to the bacterial surface in an ordered fashion, without premature aggregation, has been a major challenge in microbiology. In particular, the involvement of periplasmic chaperones in the assembly pathway has been well established. The chaperones prevent mis-aggregation and precipitation of newly synthesized pilus subunits by plucking them from the cytoplasmic membrane, assisting their correct folding and escorting them to the outer membrane where ‘usher’
601
pore-forming proteins provide a platform for assembly and export. The first two crystal structures of chaperone–pilus complexes have now been reported and have proved to be enormously rewarding. Knight and colleagues explain in their review how the concepts of donor-strand complementation and donor-strand exchange, which are suggested by the molecular architecture, can help to explain both chaperone function and pilus biogenesis. In summary, the reviews in this section once again reveal the reward of interdisciplinary research in chemical biology. The merging of synthetic organic chemistry with biology expands the toolbox for the investigation of biopolymer structure and function in molecular detail, thereby facilitating our understanding of complex biological processes. One of the major goals of the approaches highlighted here is using synthetic or semi-synthetic components as molecular flashlights to gain insight into cellular processes that cannot be fully studied with the existing methodologies of structural biology, cell biology and biochemistry. The technological advances derived from this knowledge will increasingly enable the application of biopolymers for practical purposes both in vivo and ex vivo.