High-pressure gel loader for capillary array electrophoresis microchannel plates.

Microfabricated capillary array electrophoresis (microCAE) microchannel plates are the next generation of bioanalytical separation devices. To fully exploit the capabilities of microCAE devices, supporting technology such as robotic sample loading, gel loading, microplate washing, and data analysis must be developed. Here, we describe a device for loading gel into radial capillary array electrophoresis microplates and for plate washing and drying. The microplates are locked into a loading module, and high-pressure helium is used to drive aqueous separation media or wash solutions into the microchannels through fixtures connected to the central anode reservoir. Microplates are rapidly (30 s to 5 min) loaded with separation media, such as 3%-4.8% linear polyacrylamide or 0.7%-3.0% hydroxyethyl cellulose, for electrophoresis. The effective and rapid gel-filling and plate-cleaning methods together with short electrophoretic analysis times (2-30 min) make microCAE systems versatile and powerful nucleic acid analysis platforms.

Recombinant phycobiliproteins. Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains.

A family of specific cloning vectors was constructed to express in the cyanobacterium Anabaena sp. PCC7120 recombinant C-phycocyanin subunits with one or more different tags, including the 6xHis tag, oligomerization domains, and the streptavidin-binding Strep2 tag. Such tagged alpha or beta subunits of Anabaena sp. PCC7120 C-phycocyanin formed stoichiometric complexes in vivo with appropriate wild-type subunits to give constructs with the appropriate oligomerization state and normal posttranslational modifications and with spectroscopic properties very similar to those of unmodified phycocyanin. All of these constructs were incorporated in vivo into the rod substructures of the light-harvesting complex, the phycobilisome. The C-terminal 114-residue portion of the Anabaena sp. PCC7120 biotin carboxyl carrier protein (BCCP114) was cloned and overexpressed and was biotinylated up to 20% in Escherichia coli and 40% in wild-type Anabaena sp. His-tagged phycocyanin beta--BCCP114 constructs expressed in Anabaena sp. were >30% biotinylated. In such recombinant phycocyanins equipped with stable trimerization domains, >75% of the fusion protein was specifically bound to streptavidin- or avidin-coated beads. Thus, the methods described here achieve in vivo production of stable oligomeric phycobiliprotein constructs equipped with affinity purification tags and biospecific recognition domains usable as fluorescent labels without further chemical manipulation.

Conversion of capillary electrophoresis microchip genotyping data for analysis with Genetic Profiler software.

The collection and conversion of 4-color fluorescent genotyping data from capillary array electrophoresis microchip devices and its conversion to a format easily and rapidly analyzed by Genetic Profiler genotyping software is presented. Microchip fluorescence intensity data are acquired and stored as 4-color tab-delimited text. These files are converted to electrophoretic signal data (ESD) files using a utility program (TEXT-to-ESD) written in C. TEXT-to-ESD generates an ESD file by converting text data to binary data and then appending a 632-byte ESD-file trailer. Up to 96 ESD files are then assembled into a run folder and imported into Genetic Profiler, where data are reduced to 4-color electropherograms and analyzed. In this manner, DNA fragment sizing data acquired with our high-speed electrophoretic microchip devices can be rapidly analyzed using robust commercial software. Additionally, the conversion program allows sizing of data with Genetic Profiler that have been preprocessed using other third-party software, such as BaseFinder.

Structural insights into the evolution of an antibody combining site.

The crystal structures of a germline antibody Fab fragment and its complex with hapten have been solved at 2.1 A resolution. These structures are compared with the corresponding crystal structures of the affinity-matured antibody, 48G7, which has a 30,000 times higher affinity for hapten as a result of nine replacement somatic mutations. Significant changes in the configuration of the combining site occur upon binding of hapten to the germline antibody, whereas hapten binds to the mature antibody by a lock-and-key fit mechanism. The reorganization of the combining site that was nucleated by hapten binding is further optimized by somatic mutations that occur up to 15 from bound hapten. These results suggest that the binding potential of the primary antibody repertoire may be significantly expanded by the ability of germline antibodies to adopt more than one combining-site configuration, with both antigen binding and somatic mutation stabilizing the configuration with optimal hapten complementarity.

Crystal structures of the free and liganded form of an esterolytic catalytic antibody.

The crystal structure of the esterase catalytic antibody 48G7 has been determined in the presence of hapten at 2.0 A resolution and in the absence of hapten at 2.7 A resolution. The root-mean-square difference between the two structures is 0.6 A for the variable domain and 0.7 A for the constant domain. Comparison of the active site shows that no significant changes occur upon hapten binding as main-chain and side-chain displacements are negligible. Complex formation occurs as hapten fits into a pre-formed pocket about 10 A deep. Although 151 water molecules were modeled into the 48G7-hapten structure, none are bound in the active site. Comparison of the 48G7 structures with those of other published ester hydrolysis antibodies illustrates an emerging theme used by esterolytic antibodies in binding their (nitro-)phenyl haptens and in hydrolysing their cognate esters and carbonates: hapten is bound with the aryl end buried deep in the binding pocket, and the phosphonate moiety is responsible for the majority of the binding energy to the antibody-hapten interaction.

The immunological evolution of catalysis.

The germline genes used by the mouse to generate the esterolytic antibody 48G7 were cloned and expressed in an effort to increase our understanding of the detailed molecular mechanisms by which the immune system evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transition state analogue by a factor of 10(4), primarily the result of a decrease in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with substrate, k(cat)/k(m), from 1.7 x 10(2)M(-1) min(-1) to 1.4 x 10(4)M(-1) min(-1). The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that one of the nine residues in which somatic mutations have been fixed directly contact the hapten. Thus, in the case of 48G7, affinity maturation appears to play a conformational role, either in reorganizing the active site geometry of limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic and directed active site mutants underscore the role of transition state stabilization in the evolution of this catalytic antibody.

Cryptomonad biliproteins: Bilin types and locations.

Two crytophycean phycocyanins (Cr-PCs), Hemiselmis strain HP9001 Cr-PC 612 and Falcomonas daucoides Cr-PC 69 were purified and characterized with respect to bilin numbers, types and locations. Each biliprotein carried one bilin on the α subunit and three on the ß subunit. Cr-PC 612 carried phycocyanobilin at α-Cys-18, ß-Cys-82, and ß-Cys-158, and a doubly-linked 15,16-dihydrobiliverdin at ß-DiCys-50,61. Cr-PC 569 carried phycocyanobilin at α-Cys-18 and ß-Cys-82, a singly-linked Bilin 584 at ß-Cys-158, and a doubly-linked Bilin 584 at ß-DiCys-50,61. This work, in conjunction with earlier studies on Cr-PE 545, Cr-PE 555, Cr-PE 566, and Cr-PC 645, shows that there is no conserved location for the bilin with longest wavelength visible absorption band among these proteins, and, consequently, that there is no conserved energy transfer pathway common to all native cryptophycean biliproteins. Only phycocyanobilin or phycoerythrobilin is found at ß-Cys-82; there is greater bilin variability at the other three attachment sites.

Cryptomonad biliproteins - an evolutionary perspective.

Each cryptomonad strain contains only a single spectroscopic type of biliprotein. These biliproteins are isolated as ≈50000 kDa αα'ß2 complexes which carry one bilin on the α and three on the ß subunit. Six different bilins are present on the cryptomonad biliproteins, two of which (phycocyanobilin and phycoerythrobilin) also occur in cyanobacterial and rhodophytan biliproteins, while four are known only in the cryptomonads. The ß subunit is encoded on the chloroplast genome, whereas the α subunits are encoded by a small nuclear multigene family. The ß subunits of all cryptomonad biliproteins, regardless of spectroscopic type, have highly conserved amino acid sequences, which show > 80% identity with those of rhodophytan phycoerythrin ß subunits. In contrast, cyanobacteria and red algal chloroplasts each contain several spectroscopically distinct biliproteins organized into macromolecular complexes (phycobilisomes). The data on biliproteins, as well as several other lines of evidence, indicate that the cryptomonad biliprotein antenna system is 'primitive' and antedates that of the cyanobacteria. It is proposed that the gene encoding the cryptomonad biliprotein ß subunit is the ancestral gene of the gene family encoding cyanobacterial and rhodophytan biliprotein α and ß subunits.

Phycobilins of cryptophycean algae. Novel linkage of dihydrobiliverdin in a phycoerythrin 555 and a phycocyanin 645.

Cryptomonad strain IVF2 phycoerythrin 555 carries phycoerythrobilins attached through single thioether bonds at alpha-Cys-18, beta-Cys-82, and beta-Cys-158 and a doubly linked 15,16-dihydrobiliverdin (DBV) at beta-DiCys-50,61 (for sequence numbering, see Sidler, W., Nutt, H., Kumpf, B., Frank, G., Suter, F., Brenzel, A., Wehrmeyer, W., and Zuber, H. (1990) Biol. Chem. Hoppe-Seyler 371, 573-547). Analysis of the beta-DiCys-50,61-linked DBV by 1H homonuclear and 1H-13C heteronuclear NMR spectroscopy establishes that the thioether bond from Cys-50 is to the 3"-carbon of the DBV ring A and that from Cys-61 is to the 18'-carbon of ring D, i.e. the peptide-linked bilin is an 8,12-bis(2-carboxyethyl)-3-(2-(cysteinyl-S)-ethyl)-18-(1-(cysteinyl-S)-e thyl)- 2,7,13,17-tetramethylbiladiene-ab-1,19(16H,21H)-dione. DBV is also present at beta-DiCys-50,61 in cryptomonad strain UW374 phycocyanin 645 (Wedemayer, G. J., Kidd, D. G., Wemmer, D. E., and Glazer, A. N. (1992) J. Biol. Chem. 267, 7315-7331). NMR spectroscopy shows that the thioether bonds to this DBV are also at 3" and 18'. Linkage of tetrapyrroles to polypeptides through the 3"-carbon has not hitherto been reported.

Phycobilins of cryptophycean algae. Occurrence of dihydrobiliverdin and mesobiliverdin in cryptomonad biliproteins.

Structures of the open-chain tetrapyrrole (bilin) prosthetic groups of the cryptophycean biliproteins phycocyanin 645 (Cr-PC 645; from strain UW374), phycoerythrin 566 (Cr-PE 566; from strain Bermani) and phycoerythrin 545 (Cr-PE 545; from Proteomonas sulcata Hill & Wetherbee) were examined by absorption, 1H NMR spectroscopy, and mass spectrometry. These biliproteins carry the following covalently attached bilins: Cr-PC 645 (alpha subunit) has one mesobiliverdin, (beta subunit), two phycocyanobilins and a doubly linked 15,16-dihydrobiliverdin; Cr-PC 566 (alpha), bilin 584, (beta), phycoerythrobilin and two bilin 584 chromophores (Wedemayer, G.J., Wemmer, D.E., and Glazer, A.N. (1991) J. Biol. Chem. 266, 4731-4741); Cr-PE 545 (alpha) has one 15,16-dihydrobiliverdin and (beta), only phycoerythrobilins. This is the first report of naturally occurring biliproteins carrying either 15,16-dihydrobiliverdin or mesobiliverdin chromophores. Native cryptomonad phycobiliproteins have been classified on the basis of the position of their long wavelength absorption maxima. However, comparison of the bilins of Cr-PE 566 from strain Bermani with those of Cr-PE 566 of strain CBD shows that the two proteins carry different bilins on the alpha subunit. Consequently, the identity of the bilin prosthetic groups on cryptophycean phycobiliproteins cannot be unambiguously inferred from simple inspection of the visible absorption spectra.

Phycobilins of cryptophycean algae. Structures of novel bilins with acryloyl substituents from phycoerythrin 566.

Cryptomonad strain CBD phycoerythrin 566 carries four open-chain tetrapyrrole (bilin) prosthetic groups: three singly thioether-linked bilins at alpha-19, beta-82, and beta-158 and a bilin linked through two thioether bonds at beta-50,61 (amino acid sequence numbering from Wilbanks, S. M., Wedemayer, G.J., and Glazer, A.N. (1989) J. Biol. Chem. 264, 17860-17867). The structures of all four peptide-linked prosthetic groups were determined by 1H NMR spectroscopy. The bilin at beta-82 was identified as phycoerythrobilin (PEB), a common prosthetic group in cyanobacterial and red algal phycobiliproteins. The structures of the remaining bilins were novel. The bilin at alpha-19, designated Cys-bilin 618, differed from PEB in having additional double bonds between C-2 and C-3 of ring A and between C-12' and C-12", i.e. an acryloyl substituent at C-12 of ring C. The doubly linked bilin at beta-50,61 designated DiCys-bilin 584, differed from doubly linked PEB (Schoenleber, R.W., Lundell, D.J., Glazer, A.N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5481-5484) in possessing an acryloyl substituent at C-12 of ring C in place of a propionyl substituent. Similarly, the bilin at beta-158, designated Cys-bilin 584, differed from singly-linked PEB in possessing an acryloyl substituent at C-12 of ring C in place of a propionyl substituent. The three novel cryptomonad bilins join heme d1 and chlorophylls c1, c2, and c3 as the only known porphyrin-derived natural products with acryloyl substituents.

Posttranslational modifications of the beta subunit of a cryptomonad phycoerythrin. Sites of bilin attachment and asparagine methylation.

Determination of the partial amino acid sequence of the beta subunit of cryptomonad strain CBD phycoerythrin 566 established the nature, locations, and modes of attachment of the three bilin prosthetic groups and revealed a site of posttranslational methylation. Isolation of peptides cross-linked by a phycobiliviolin led to an unambiguous assignment of two thioether linkages, from residues beta-Cys-50 and beta-Cys-61 to this bilin. Two bilins were attached through single thioether linkages, a phycobiliviolin at beta-Cys-158 and a phycoerythrobilin at beta-Cys-82 (the residue numbering is that for B-phycoerythrin; Sidler, W., Kumpf, B., Suter, F., Morisset, W., Wehrmeyer, W., and Zuber, H. (1985) Biol. Chem. Hoppe-Seyler 366, 233-244). The partial sequences (99 residues) established for phycoerythrin 566 beta subunit showed a 79% identity with that of the red algal Porphyridium cruentum B-phycoerythrin beta subunit. A particularly remarkable finding is that the unique methylasparagine residue at position beta-72, highly conserved in cyanobacterial and red algal phycobiliproteins (Klotz, A. V., and Glazer, A. N. (1987) J. Biol. Chem. 262, 17350-17355), is also present at beta-72 in the cryptomonad phycoerythrin. Comparison of the locations of donor and acceptor bilins in cryptomonad phycoerythrin with those found for cyanobacterial and red algal phycobiliproteins showed different favored pathways of energy migration in the cryptomonad protein.

Dinoflagellate with blue-green chloroplasts derived from an endosymbiotic eukaryote.

The dinoflagellate, Amphidinium wigrense, contains triple membrane-bound bodies we have termed "blue-green chloroplasts." We believe these chloroplasts were derived from a cryptomonad endosymbiont similar to that present in another blue-green dinoflagellate, Gymnodinium acidotum. These dinoflagellates provide evidence that a chloroplast has evolved from an endosymbiotic eukaryote.