Evolutionary analysis of the multigene pregnancy-specific β1-Glycoprotein family: Separation of historical and nonhistorical signals

The pregnancy-specific ₁-glycoproteins (PSG) form a large family of closely related proteins. Using newly developed methods of sequence analysis, in combination with protein modeling, we provide a framework for investigating the evolution and biological function of genes like the PSG. Evolutionary trees, based on C-terminal sequence, group PSG genes in a manner consistent with their genomic organization. Trees constructed using the N-terminal domain sequences are unreliable as an indicator of phylogeny because of non-neutral processes of sequence change. During duplication of the PSG genes, evolutionary pressures have resulted in a gradient of constrained change across each gene. The N-terminal domains show a nonrandom pattern of amino acid substitutions clustered in the immunoglobulin complementarity-determining region (CDR)-like regions, which appear to be important in the function of the protein.     

Modeling the temperature response of nitrification

To model nitrification rates in soils, it is necessary to have equations that accurately describe the effect of environmental variables on nitrification rates. A variety of equations have been used previously to describe the effect of temperature on rates of microbial processes. It is not clear which of these best describes the influence of temperature on nitrification rates in soil. I compared five equations for describing the effects of temperature on nitrification in two soils with very different temperature optima from a California oak woodland-annual grassland. The most appropriate equation depended on the range of temperatures being evaluated. A generalized Poisson density function best described temperature effects on nitrification rates in both soils over the range of 5 to 50 °C; however, the Arrhenius equation best described temperature effects over the narrower range of soil temperatures that normally occurs in the ecosystem (5 to 28 °C). Temperature optima for nitrification in most of the soils were greater than even the highest soil temperatures recorded at the sites. A model accounting for increased maintenance energy requirements at higher temperatures demonstrates how net energy production, rather than the gross energy production from nitrification, is maximized during adaptation by nitrifier populations to soil temperatures.     

Hybridomas in a bioreactor cascade: modeling and determination of growth and death kinetics

Hybridomas were cultured under steady-state conditions in a series of two continuous stirred-tank reactors (CSTRs), using a serum-free medium. The substrate not completely converted in the first CSTR, was transported with the cells to the second one and very low growth rates, high death rates, and lysis of viable cells were observed in this second CSTR. These conditions are hardly accessible in a single vessel, because such experiments would be extremely time-consuming and unstable due to a low viability. In contrast to what is often observed in literature, kinetic parameters could thus be derived without the neccessity for extrapolation to lower growth rates. Good agreement with literature averages for other hybridomas was found. Furthermore, showing that the reactor series is a valuable research tool for kinetic studies under extreme conditions, the possibility to observe cell death under stable and defined steady-state conditions offers interesting opportunities to investigate apoptosis and necrosis. Additionally, a model was developed that describes hybridoma growth and monoclonal antibody production in the bioreactor cascade on the basis of glutamine metabolism. Good agreement between the model and the experiments was found.Abbreviation MAb Monoclonal antibody Nomenclature C _AConcentration of any (mol m^-3) component A D Dilution rate (s^-1) K _dDeath-rate constant (mol m^-3) K _lLysis-rate constant (mol m^-3) K _sMonod constant (mol m^-3) m Maintenance coefficient (mol cell^-1 s^-1) q Specific consumption (mol cell^-1 s^-1) or production rate t Time (s) X Cell concentration (cell m^-3) Y Yield coefficient (cell mol^-1) Greek symbols _d Specific death rate (s^-1) _l Specific lysis rate (s^-1) of viable cells _net Net specific growth (s^-1) rate _true True specific growth (s^-1) rate     

A comparison of the predicted and X-ray structures of angiogenin. Implications for further studies of model building of homologous proteins

The three-dimensional structure of human angiogenin has been determined by X-ray crystallography and is compared here with an earlier model which predicted its structure, based on the homology of angiogenin with bovine pancreatic ribonuclease A. Comparison of the predicted model and crystal structure shows that the active-site histidine residues and the core of the angiogenin molecule, including most of the-strands and-helices, were predicted reasonably well. However, the structure of the surface loop regions and residues near the truncated C-terminus differs significantly. The C-terminal segment includes the active-site residues Asp-116, Gln-117, and Ser-118; Gln-117 in particular has been shown to be important in affecting the ribonucleolytic activity of angiogenin. Also, the orientation of one helix in the model differed from the orientation observed experimentally by about 20°, resulting in a large displacement of this chain segment. The difficulty encountered in predicting the surface loop regions has led to a new algorithm [Palmer and Scheraga (1991),J. Comput. Chem.,12, 505–526; (1992),J. Comput. Chem.,13, 329–350] for predicting the conformations of surface loops.     

Quantitative in vivo nuclear magnetic resonance studies of hybridoma metabolism

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of a murine hybridoma cell line at two feed glutamine concentrations, 4.0 and 1.7 mM. Carbon-13 labeling patterns were used in conjunction with nutrient uptake rates to calculate the metabolic fluxes through the glycolytic pathway, the pentose shunt, the malate shunt, lipid biosynthesis, and the tricarboxylic acid (TCA) cycle. Decreasing the feed glutamine concentration significantly decreased glutamine uptake but had little effect on glucose metabolism. A significant incrase in antibody productivity occurred upon decreasing the feed glutamine level. The increased antibody productivity in concert with decreased glutamine uptake and no apparent change in glucolytic metabolism suggests that antibody production was not energy limited. Metabolic flux calculations indicate that (1) approximately 92% of the glucose consumed proceeds directly through glycolysis with 8% channeled through the pentose shunt; (2) lipid biosynthesis appears to be greater than malate shunt activity; and (3) considerable exchange occurs between TCA cycle intermediates and amino acid metabolic pools, leading to substantial loss of (13)C label from the TCA cycle. These results illustrate that (13)NMR spectroscopy is a powerfulf tool in the calculation of metabolic fluxes, particularly for exchange pathways where no net flux occurs. (c) 1994 John Wiley & Sons, Inc.     

Conservation of polyproline II helices in homologous proteins: implications for structure prediction by model building.

Left-handed polyproline II (PPII) helices commonly occur in globular proteins in segments of 4-8 residues. This paper analyzes the structural conservation of PPII-helices in 3 protein families: serine proteinases, aspartic proteinases, and immunoglobulin constant domains. Calculations of the number of conserved segments based on structural alignment of homologous molecules yielded similar results for the PPII-helices, the alpha-helices, and the beta-strands. The PPII-helices are consistently conserved at the level of 100-80% in the proteins with sequence identity above 20% and RMS deviation of structure alignments below 3.0 A. The most structurally important PPII segments are conserved below this level of sequence identity. These results suggest that the PPII-helices, in addition to the other 2 secondary structure classes, should be identified as part of structurally conserved regions in proteins. This is supported by similar values for the local RMS deviations of the aligned segments for the structural classes of PPII-helices, alpha-helices, and beta-strands. The PPII-helices are shown to participate in supersecondary elements such as PPII-helix/alpha-helix. The conservation of PPII-helices depends on the conservation of a supersecondary element as a whole. PPII-helices also form links, possibly flexible, in the interdomain regions. The role of the PPII-helices in model building by homology is 2-fold; they serve as additional conserved elements in the structure allowing improvement of the accuracy of a model and provide correct chain geometry for modeling of the segments equivalenced to them in a target sequence. The improvement in model building is demonstrated in 2 test studies.     

Homology models of two isozymes of manganese peroxidase: Prediction of a Mn(II) binding site

The three-dimensional structures of two isozymes of manganese peroxidase (MnP) have been predicted from homology modeling using lignin peroxidase as a template. Although highly homologous, MnP differs from LiP by the requirement of Mn(II) as an intermediate in its oxidation of substrates. The Mn(II) site is absent in LiP and unique to the MnP family of peroxidases. The model structures were used to identify the unique Mn(II) binding sites, to determine to what extent they were conserved in the two isozymes, and to provide insight into why this site is absent in LiP. For each isozyme of MnP, three candidate Mn(II) binding sites were identified. Energy optimizations of the three possible Mn(II) enzyme complexes allowed the selection of the most favorable Mn(II) binding site as one with the most anionic oxygen moieties best configured to act as ligands for the Mn(II). At the preferred site, the Mn(II) is coordinated to the carboxyl oxygens of Glu-35, Glu-39, and Asp-179, and a propionate group of the heme. The predicted Mn(II) binding site is conserved in both isozymes. Comparison between the residues at this site in MnP and the corresponding residues in LiP shows that two of the three anionic residues in MnP are replaced by neutral residues in LiP, explaining why LiP does not bind Mn(II). © 1994 Wiley-Liss, Inc.     

Interaction of the FAFB-containing subunit with the Photosystem 1 core heterodimer

The structure of the predicted amino acid sequence in the F_X domain of Photosystem 1 was studied by molecular modeling and a working hypothesis was developed for the functional interaction of PsaC with the core heterodimer. We propose that the intervening sequences between homologous cysteines in the F_X cluster form two flexible loops and participate in the binding of PsaC, and that the arginine residues in the two surface-exposed loops may promote the interaction between the P₇₀₀–F_X core and the subunit. The model was tested experimentally; chemical modification of arginine residues in the P₇₀₀–F_X core using phenylglyoxal prevented reconstitution of the core with PsaC and PsaD after insertion of FeS clusters in vitro. Treatment of the P₇₀₀–F_X core with trypsin also prevented reconstitution of terminal electron transfer to F_AF_B, although neither treatments affected the electron transfer to F_X as judged by flash kinetic spectrophotometry. Electron transfer in the P₇₀₀–F_AF_B complex was not impaired by either phenylglyoxal or trypsin treatment indicating that the small subunit(s) protect the arginine residues that become chemically modified or cleaved. The data are consistent with the working model and point to additional experiments designed to identify the specific residues involved in the interaction between the P₇₀₀–F_X core and PsaC.Abbreviations PG- phenylglyoxal - PS 1- Photosystem 1