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Semisynthology—a pathway to probing protein function
TIBS - September 1980
XXVlII
Semisynthology - a pathway to probing protein function Semisynthetic Proteins
by R. E. Offord, John Wiley and Sons Ltd, Chichester, New York, Brisbane and Toronto, 1980.s (xi + 235 pages) ISBN 0 471 27615 4 This text represents the first handbook devoted exclusively to the methods of protein semisynthesis, in which 'fragments of naturally occurring proteins are used as ready-made intermediates in the construction of proteins of novel sequence'. By the type of controlled alteration of particular amino acid residues possible through synthesis, analogues of proteins can be obtained both for studies of the roles of chosen residues in biological activity and for insertion of probes for tracer studies (in the laboratory and clinically). Since total synthesis of large polypeptides still suffers from technical limitations which make it non-routine, semisynthesis offers a more viable approach at present for using peptide synthesis procedures to alter protein sequence. And, as pointed out by the author, 'If we restrict ourselves to molecules larger than insulin, there have been more protein analogues produced by semisynthesis than by traditional methods'. This assertion notwithstanding, it seems apparent that at least one intent of the author, and with good reason, is to show that semisynthesis should in fact become a 'traditional method'. The primary areas covered by the text comprise the chemical techniques of semisynthesis and published results, with an overwhelming emphasis on the former. All major aspects of preparation of semisynthetic proteins are discussed. Three chapters are devoted to fragmentation, including protection before cleavage, cleavage and separation of fragments, and protection after cleavage. Two chapters are devoted to synthesis and modification of fragments, and two others to reconstitution of native and synthetic components (one on coupling and a second on deprotection and purification of products). In addition, one of two appendices is on methods of characterization of products and intermediates, with the second being on technical problems. For all chemical methods covered, the experimental details of several practical examples are presented in some depth. Helpful technical notes and references are included. These real-life examples provide an excellent guide to focus on ways in which the methods are applied. While centred heavily on the particular spectrum of examples from the
author's laboratory, this 'practicum' aspect of the text does benefit from the evaluative perspective gained from his familiarity With the actual experiments. Even given a reasonable state of methodological advance, the single chapter of review of published work graphically reveals that semisynthesis is still the toy of a relatively few and not the tool of a whole science. To be sure, an impressively growing list of proteins is cited as objects of productive semisynthetic manipulation. These proteins include insulin, bovine pancreatic ribonuclease, myoglobin, cytochrome c, staphylococcal nuclease, phospholipase A2, somatotropin, immunoglobulin, ferredoxins, trypsin, and protease inhibitors. None the less, this list represents only a small proportion of proteins of known sequence. Given clear cut advantages which make sequence variation by semisynthesis more controlled and therein more appealing than that by such approaches as traditional chemical modification, mutation, and comparison of evolutionarily variant sequences, the potential level of use is far greater than pre-
sent achievement. Some of thelimiting factors, such as problems of solubility of protected fragments and the somewhat obstinate nature of many fragment conden. sations, become apparent in the text. But such technical problems are surmountable. One suspects, more generally, that the comprehensive use of semisynthesis by protein chemists and biochemists, who have proteins in hand and are looking around for ways to study them, will have to await an awareness that the dividends (the information that can be gained) are at least a reasonable cause for enduring the technical barriers. Whilethe text of Semisynthetic Proteins doesn't emphasize what biochemical information has been Obtained for the protein cases studied, it does present an excellent opportun!ty for potential users to see what techniques are required to make semisynthetic species and what systems have proven amenable so far. The book also offers a chance for those in the field to review current technological developments. IRWIN M. CttAIKEN
Irwin M. Chaiken is at the Laboratory of Chemical Biology, NIAMDD National Institutes of Health, Bethesda, Maryland20205, U.S.A.
Valuable plant book would benefit from pruning Plant M e m b r a n e Transport: Current Conceptual Issues
edited by R. M. Spanswick, IV. J. Lucas and J. Dainty. Elsevier/North-Holland Bio'medical Press, Amsterdam, New York and London, 1980. $73.251Dfl. 150.00 (xviii + 670pages) ISBN 0 444 80192 8 This book reports the proceedings of the International Workshop on Membrane Transport in Plants which was held in Toronto in July 1979. This was the fifth in a continuing series of such gatherings which have been held since 1968. The proceedings of two of the previous Workshops (Ion Transport in Plants, edited by W. P. Anderson, Academic Press, 1973; Membrane Transport in Plants, edited by U. Zimmerman and J. Dainty, SpringerVerlag, 1974) were important contributions to the literature on transport phenomena in plants and I suspect that this latest addition to the series will prove to be another milestone. The relatively short lag between conference and publication has ensured that the book has an immediate relevance to current research and all concerned with its production (editors, contributors and publisher) deserve congratulations for their efforts. The contributions in the book are of two
types. The first 390 pages are occupied by 25 papers, each 8--10 pages in length, which were the invited contributions of the Workshop. Each paper is followed by a report of the ensuing discussion. These papers, which are all written by accepted authorities in the field, are grouped into seven sections each dealing with a different aspect of transport in plants. The seven sections are: ionic relations of selected plant systems; water and osmoregulation; chemiosmosis and ATPases in membrane transport; co-transport of sugars and amino acids; membrane transport in Characeae; action potentials in plants; and possible roles for membrane transport in disease and development. The other contributions are two page abstracts of papers presented in poster sessions. There are 125 of these and they occupy the last 250 pages of the book. As can be seen from the above list the range of topics covered is very wide and anyone with an interest in the mechanisms of solute and water transport in plants should find something of relevance in the book. The majority of the papers are concemed with transport across the plasmalemma and tonoplast and those with an interest in transport in other organelles, transport in the xylem or in the integration
XXVlII
Semisynthology - a pathway to probing protein function Semisynthetic Proteins
by R. E. Offord, John Wiley and Sons Ltd, Chichester, New York, Brisbane and Toronto, 1980.s (xi + 235 pages) ISBN 0 471 27615 4 This text represents the first handbook devoted exclusively to the methods of protein semisynthesis, in which 'fragments of naturally occurring proteins are used as ready-made intermediates in the construction of proteins of novel sequence'. By the type of controlled alteration of particular amino acid residues possible through synthesis, analogues of proteins can be obtained both for studies of the roles of chosen residues in biological activity and for insertion of probes for tracer studies (in the laboratory and clinically). Since total synthesis of large polypeptides still suffers from technical limitations which make it non-routine, semisynthesis offers a more viable approach at present for using peptide synthesis procedures to alter protein sequence. And, as pointed out by the author, 'If we restrict ourselves to molecules larger than insulin, there have been more protein analogues produced by semisynthesis than by traditional methods'. This assertion notwithstanding, it seems apparent that at least one intent of the author, and with good reason, is to show that semisynthesis should in fact become a 'traditional method'. The primary areas covered by the text comprise the chemical techniques of semisynthesis and published results, with an overwhelming emphasis on the former. All major aspects of preparation of semisynthetic proteins are discussed. Three chapters are devoted to fragmentation, including protection before cleavage, cleavage and separation of fragments, and protection after cleavage. Two chapters are devoted to synthesis and modification of fragments, and two others to reconstitution of native and synthetic components (one on coupling and a second on deprotection and purification of products). In addition, one of two appendices is on methods of characterization of products and intermediates, with the second being on technical problems. For all chemical methods covered, the experimental details of several practical examples are presented in some depth. Helpful technical notes and references are included. These real-life examples provide an excellent guide to focus on ways in which the methods are applied. While centred heavily on the particular spectrum of examples from the
author's laboratory, this 'practicum' aspect of the text does benefit from the evaluative perspective gained from his familiarity With the actual experiments. Even given a reasonable state of methodological advance, the single chapter of review of published work graphically reveals that semisynthesis is still the toy of a relatively few and not the tool of a whole science. To be sure, an impressively growing list of proteins is cited as objects of productive semisynthetic manipulation. These proteins include insulin, bovine pancreatic ribonuclease, myoglobin, cytochrome c, staphylococcal nuclease, phospholipase A2, somatotropin, immunoglobulin, ferredoxins, trypsin, and protease inhibitors. None the less, this list represents only a small proportion of proteins of known sequence. Given clear cut advantages which make sequence variation by semisynthesis more controlled and therein more appealing than that by such approaches as traditional chemical modification, mutation, and comparison of evolutionarily variant sequences, the potential level of use is far greater than pre-
sent achievement. Some of thelimiting factors, such as problems of solubility of protected fragments and the somewhat obstinate nature of many fragment conden. sations, become apparent in the text. But such technical problems are surmountable. One suspects, more generally, that the comprehensive use of semisynthesis by protein chemists and biochemists, who have proteins in hand and are looking around for ways to study them, will have to await an awareness that the dividends (the information that can be gained) are at least a reasonable cause for enduring the technical barriers. Whilethe text of Semisynthetic Proteins doesn't emphasize what biochemical information has been Obtained for the protein cases studied, it does present an excellent opportun!ty for potential users to see what techniques are required to make semisynthetic species and what systems have proven amenable so far. The book also offers a chance for those in the field to review current technological developments. IRWIN M. CttAIKEN
Irwin M. Chaiken is at the Laboratory of Chemical Biology, NIAMDD National Institutes of Health, Bethesda, Maryland20205, U.S.A.
Valuable plant book would benefit from pruning Plant M e m b r a n e Transport: Current Conceptual Issues
edited by R. M. Spanswick, IV. J. Lucas and J. Dainty. Elsevier/North-Holland Bio'medical Press, Amsterdam, New York and London, 1980. $73.251Dfl. 150.00 (xviii + 670pages) ISBN 0 444 80192 8 This book reports the proceedings of the International Workshop on Membrane Transport in Plants which was held in Toronto in July 1979. This was the fifth in a continuing series of such gatherings which have been held since 1968. The proceedings of two of the previous Workshops (Ion Transport in Plants, edited by W. P. Anderson, Academic Press, 1973; Membrane Transport in Plants, edited by U. Zimmerman and J. Dainty, SpringerVerlag, 1974) were important contributions to the literature on transport phenomena in plants and I suspect that this latest addition to the series will prove to be another milestone. The relatively short lag between conference and publication has ensured that the book has an immediate relevance to current research and all concerned with its production (editors, contributors and publisher) deserve congratulations for their efforts. The contributions in the book are of two
types. The first 390 pages are occupied by 25 papers, each 8--10 pages in length, which were the invited contributions of the Workshop. Each paper is followed by a report of the ensuing discussion. These papers, which are all written by accepted authorities in the field, are grouped into seven sections each dealing with a different aspect of transport in plants. The seven sections are: ionic relations of selected plant systems; water and osmoregulation; chemiosmosis and ATPases in membrane transport; co-transport of sugars and amino acids; membrane transport in Characeae; action potentials in plants; and possible roles for membrane transport in disease and development. The other contributions are two page abstracts of papers presented in poster sessions. There are 125 of these and they occupy the last 250 pages of the book. As can be seen from the above list the range of topics covered is very wide and anyone with an interest in the mechanisms of solute and water transport in plants should find something of relevance in the book. The majority of the papers are concemed with transport across the plasmalemma and tonoplast and those with an interest in transport in other organelles, transport in the xylem or in the integration