Gary J. Wedemayer

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 104, 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, kcat/Km, from 1.7 × 102 M−1 min−1 to 1.4 × 104 M−1 min−1. The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that none 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 or 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.

Finding important sites in protein sequences

By using sequence information from an aligned protein family, a procedure is exhibited for finding sites that may be functionally or structurally critical to the protein. Features based on sequence conservation within subfamilies in the alignment and associations between sites are used to select the sites. The sites are subject to statistical evaluation correcting for phylogenetic bias in the collection of sequences. This method is applied to two families: the phycobiliproteins, light-harvesting proteins in cyanobacteria, red algae, and cryptomonads, and the globins that function in oxygen storage and transport. The sites identified by the procedure are located in key structural positions and merit further experimental study.

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 β subunit. Cr-PC 612 carried phycocyanobilin at α-Cys-18, β-Cys-82, and β-Cys-158, and a doubly-linked 15,16-dihydrobiliverdin at β-DiCys-50,61. Cr-PC 569 carried phycocyanobilin at α-Cys-18 and β-Cys-82, a singly-linked Bilin 584 at β-Cys-158, and a doubly-linked Bilin 584 at β-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 β-Cys-82; there is greater bilin variability at the other three attachment sites.

PHAGOTROPHY IN THE FRESHWATER, PHOTOSYNTHETIC DINOFLAGELLATE AMPHIDINIUM CRYOPHILUM1

The cold‐water, photosynthetic dinoflagellate Amphidinium cryophilum Wedemayer, Wilcox & Graham feeds phagotrophically on other dinoflagellate species. Food is ingested through a feeding tube, termed here the “phagopod,” which extends from the antapex. The peduncle of this organism plays no observable role in the feeding process. The phagopod is essentially a hollow cylinder composed electron‐opaque material that is possibly deposited on a membrane. No Amphidinium cytoplasmic components, including microtubules or other cytoskeletal elements, were observed in the phagopod. Prefeeding cells aggregate, in small clumps near prey organisms with their phagopods extended. Eventually some cells commence feeding, first inserting the phagopod through the prey cell‐covering and then slowly, over a period of 10 min or more, drawing cytoplasm through the phagopod and into a nascent food vacuole. Both light and electron microscopy suggest that one or more prey cell amphiesmal membranes remain intact during the feeding process. Upon completion of feeding, the Amphidinium cell swims off with a prominent food vacuole in the hypocone, leaving at least part of the phagopod attached to the prey cell. Phagotrophy in A. cryophilum seems to vary with light intensity. At low light intensities, cells feed phagotrophically and are nearly colorless, whereas at high light levels they feed much less frequently, if at all, and are brightly pigmented.