Wed, 20 Aug 2008 22:20:32 BST CiteULike: stevanspringer combinatorial http://www.citeulike.org/user/stevanspringer/tag/combinatorial CiteULike.org en-gb Copyright © 2004-2008 citeulike.org http://www.citeulike.org/user/stevanspringer/article/2968263 <i>International Journal of Developmental Biology, Vol. 52 (2008), pp. 759-768.</i><br /><br />Sperm of the oyster, Crassostrea gigas, have ring-shaped acrosomes that, after exocytosis, bind the sperm to the egg vitelline layer. Isolated acrosomal rings contain proteins of various sizes: 35-, 48-, 63-, 75- and 88-kDa. These proteins, called bindins, have identical 24-residue signal peptides and conserved 97-residue N-terminal sequences, and they differ in mass because of the presence of between 1 and 5 tandemly repeated 134-residue fucose-binding lectin (F-lectin) domains. Southern blots suggest that oyster bindin is a single copy gene, but F-lectin repeat number and sequence are variable within and between individuals. Eight residues in the F-lectin fucose-binding groove are subject to positive diversifying selection, indicating a history of adaptive evolution at the lectin's active site. There is one intron in the middle of each F-lectin repeat, and recombination in this intron creates many combinations of repeat halves. Alternative splicing creates many additional size and sequence variants of the repeat array. Males contain full-length bindin cDNAs of all 5 possible sizes, but only one or two protein mass forms exist in each individual. Sequence analysis indicates that recombination and alternate splicing create hundreds, possibly thousands, of different bindin sequences in C. gigas. The extreme within-species sequence variation in the F-lectin sequence of oyster bindin is a novel finding; most male gamete-recognition proteins are much less variable. In experimental conditions oyster eggs have poor polyspermy blocks, and bindin diversity could be an evolutionary response by sperm to match egg receptors that have diversified to avoid being fertilized by multiple sperm. Oyster sperm bindin is a combinatorial fucose lectin with remarkable intra-species diversity Stevan Springer Gary Moy Daniel Friend Willie Swanson Victor Vacquier International Journal of Developmental Biology, Vol. 52 (2008), pp. 759-768. 2008-07-07T03:41:38-00:00 2008 International Journal of Developmental Biology 52 759 768 combinatorial crassostrea gamete_recognition polymorphism http://www.citeulike.org/user/stevanspringer/article/2710268 <i></i> Evolution on a chip : Nature News 2008-04-23T21:25:42-00:00 adaptation combinatorial news http://www.citeulike.org/user/stevanspringer/article/1569218 <i>Nature, Vol. 448, No. 7155., pp. 828-831.</i> Selection and evolution of enzymes from a partially randomized non-catalytic scaffold Burckhard Seelig Jack Szostak doi:10.1038/nature06032 Nature, Vol. 448, No. 7155., pp. 828-831. 2007-08-16T13:35:51-00:00 Nature 0028-0836 448 7155 828 831 Nature Publishing Group adaptation combinatorial protein_structure http://www.citeulike.org/user/stevanspringer/article/1223151 <i>J. Biol. Chem., Vol. 281, No. 31. (4 August 2006), pp. 22378-22385.</i><br /><br />A novel, quantitative saturation (QS) scanning strategy was developed to obtain a comprehensive data base of the structural and functional effects of all possible mutations across a large protein-protein interface. The QS scan approach was applied to the high affinity site of human growth hormone (hGH) for binding to its receptor (hGHR). Although the published structure-function data base describing this system is probably the most extensive for any large protein-protein interface, it is nonetheless too sparse to accurately describe the nature of the energetics governing the interaction. Our comprehensive data base affords a complete view of the binding site and provides important new insights into the general principles underlying protein-protein interactions. The hGH binding interface is highly adaptable to mutations, but the nature of the tolerated mutations challenges generally accepted views about the evolutionary and biophysical pressures governing protein-protein interactions. Many substitutions that would be considered chemically conservative are not tolerated, while conversely, many non-conservative substitutions can be accommodated. Furthermore, conservation across species is a poor predictor of the chemical character of tolerated substitutions across the interface. Numerous deviations from generally accepted expectations indicate that mutational tolerance is highly context dependent and, furthermore, cannot be predicted by our current knowledge base. The type of data produced by the comprehensive QS scan can fill the gaps in the structure-function matrix. The compilation of analogous data bases from studies of other protein-protein interactions should greatly aid the development of computational methods for explaining and designing molecular recognition. 10.1074/jbc.M603826200 Comprehensive and Quantitative Mapping of Energy Landscapes for Protein-Protein Interactions by Rapid Combinatorial Scanning Gabor Pal Jean-Louis Kouadio Dean Artis Anthony Kossiakoff Sachdev Sidhu doi:10.1074/jbc.M603826200 J. Biol. Chem., Vol. 281, No. 31. (4 August 2006), pp. 22378-22385. 2007-04-12T23:38:09-00:00 2006 J. Biol. Chem. 281 31 22378 22385 combinatorial function mutagenesis http://www.citeulike.org/user/stevanspringer/article/1223150 <i>Science, Vol. 244, No. 4908. (2 June 1989), pp. 1081-1085.</i><br /><br />A strategy, called alanine-scanning mutagenesis, was used to identify specific side chains in human growth hormone (hGH) that strongly modulate binding to the hGH receptor cloned from human liver. Single alanine mutations (62 in total) were introduced at every residue contained within the three discontinuous segments of hGH (residues 2 to 19, 54 to 74, and 167 to 191) that have been implicated in receptor recognition. The alanine scan revealed a cluster of a dozen large side chains that when mutated to alanine each showed more than a four times lower binding affinity to the hGH receptor. Many of these residues that promote binding to the hGH receptor are altered in homologs of hGH (such as placental lactogens and prolactins) that do not bind tightly to the hGH receptor. The overall folding of these mutant proteins was indistinguishable from that of the wild-type hGH, as determined by strong cross-reactivities with seven different conformationally sensitive monoclonal antibodies. The alanine scan also identified at least one side chain, Glu174, that hindered binding because when it was mutated to alanine the receptor affinity increased by more than a factor of four. 10.1126/science.2471267 High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis Bc Cunningham Ja Wells doi:10.1126/science.2471267 Science, Vol. 244, No. 4908. (2 June 1989), pp. 1081-1085. 2007-04-12T23:33:49-00:00 1989 Science 244 4908 1081 1085 combinatorial function mutagenesis http://www.citeulike.org/user/stevanspringer/article/1223141 <i>Nat Biotechnol, Vol. 17, No. 10. (October 1999), pp. 936-937.</i> Defining epitopes: It's not as easy as it seems. NS Greenspan E Di Cera doi:10.1038/13590 Nat Biotechnol, Vol. 17, No. 10. (October 1999), pp. 936-937. 2007-04-12T23:06:51-00:00 1999 Nat Biotechnol 1087-0156 17 10 936 937 combinatorial function mutagenesis protein_structure http://www.citeulike.org/user/stevanspringer/article/1222878 <i>PNAS, Vol. 101, No. 25. (22 June 2004), pp. 9205-9210.</i><br /><br />Mutagenesis of protein-encoding sequences occurs ubiquitously; it enables evolution, accumulates during aging, and is associated with disease. Many biotechnological methods exploit random mutations to evolve novel proteins. To quantitate protein tolerance to random change, it is vital to understand the probability that a random amino acid replacement will lead to a protein's functional inactivation. We define this probability as the "x factor." Here, we develop a broadly applicable approach to calculate x factors and demonstrate this method using the human DNA repair enzyme 3-methyladenine DNA glycosylase (AAG). Three gene-wide mutagenesis libraries were created, each with 105 diversity and averaging 2.2, 4.6, and 6.2 random amino acid changes per mutant. After determining the percentage of functional mutants in each library using high-stringency selection (>19,000-fold), the x factor was found to be 34% +/- 6%. Remarkably, reanalysis of data from studies of diverse proteins reveals similar inactivation probabilities. To delineate the nature of tolerated amino acid substitutions, we sequenced 244 surviving AAG mutants. The 920 tolerated substitutions were characterized by substitutability index and mapped onto the AAG primary, secondary, and known tertiary structures. Evolutionarily conserved residues show low substitutability indices. In AAG, beta strands are on average less substitutable than alpha helices; and surface loops that are not involved in DNA binding are the most substitutable. Our results are relevant to such diverse topics as applied molecular evolution, the rate of introduction of deleterious alleles into genomes in evolutionary history, and organisms' tolerance of mutational burden. 10.1073/pnas.0403255101 Protein tolerance to random amino acid change Haiwei Guo Juno Choe Lawrence Loeb doi:10.1073/pnas.0403255101 PNAS, Vol. 101, No. 25. (22 June 2004), pp. 9205-9210. 2007-04-12T20:31:15-00:00 2004 PNAS 101 25 9205 9210 combinatorial mutagenesis http://www.citeulike.org/user/stevanspringer/article/1222858 <i>PNAS, Vol. 90, No. 9. (1 May 1993), pp. 4246-4250.</i><br /><br />10.1073/pnas.90.9.4246 Additivity of Mutant Effects Assessed by Binomial Mutagenesis Lm Gregoret Rt Sauer PNAS, Vol. 90, No. 9. (1 May 1993), pp. 4246-4250. 2007-04-12T20:24:03-00:00 1993 PNAS 90 9 4246 4250 combinatorial function mutagenesis http://www.citeulike.org/user/stevanspringer/article/1156109 <i>Methods Mol Biol, Vol. 357 (2007), pp. 385-406.</i><br /><br />Combinatorial phage display technology may be applied to decipher the molecular diversity of peptide binding specificity to isolated proteins, purified antibodies, cell surfaces, intracellular/cyto-domains, and blood vessels in vivo. The application of such a strategy ranges from identifying receptor-ligand pairs and antigen binding sites to understanding the progression of diseases by their differential expression patterns and developing therapeutic targeting strategies. Different strategies can be used to isolate peptides from diverse libraries displayed on the surface of bacteriophage by exposing the library to a target molecule or organ, washing away nonbinding phage, eluting and amplifying the bound phage for multiple round use, and then analyzing the peptide sequences of the enriched phage. The following methods first outline the construction of a phage library and then delineate various in vitro and in vivo biopanning applications to probe isolated integrins, purified antibodies, cell surface molecules, and vascular endothelial cells. Techniques to decipher molecular diversity by phage display. DR Christianson MG Ozawa R Pasqualini W Arap doi:10.1385/1-59745-214-9:385 Methods Mol Biol, Vol. 357 (2007), pp. 385-406. 2007-03-12T21:08:17-00:00 2007 Methods Mol Biol 1064-3745 357 385 406 combinatorial review