A structural census of the current population of protein sequences

We examine the occurrence of the ≈300 known protein folds in different groups of organisms. To do this, we characterize a large fraction of the currently known protein sequences (≈140,000) in structural terms, by matching them to known structures via sequence comparison (or by secondary-structure class prediction for those without structural homologues). Overall, we find that an appreciable fraction of the known folds are present in each of the major groups of organisms (e.g., bacteria and eukaryotes share 156 of 275 folds), and most of the common folds are associated with many families of nonhomologous sequences (i.e., >10 sequence families for each common fold). However, different groups of organisms have characteristically distinct distributions of folds. So, for instance, some of the most common folds in vertebrates, such as globins or zinc fingers, are rare or absent in bacteria. Many of these differences in fold usage are biologically reasonable, such as the folds of metabolic enzymes being common in bacteria and those associated with extracellular transport and communication being common in animals. They also have important implications for database-based methods for fold recognition, suggesting that an unknown sequence from a plant is more likely to have a certain fold (e.g., a TIM barrel) than an unknown sequence from an animal.

Structure and function in rhodopsin: Kinetic studies of retinal binding to purified opsin mutants in defined phospholipid–detergent mixtures serve as probes of the retinal binding pocket

In the current standard procedure for preparation of mammalian rhodopsin mutants, transfected COS-1 cells expressing the mutant opsin genes are treated with 5 μM 11-cis-retinal before detergent solubilization for purification. We found that binding of 11-cis-retinal to opsin mutants with single amino acid changes at Trp-265 (W265F,Y,A) and a retinitis pigmentosa mutant (A164V) was far from complete and required much higher concentrations of 11-cis-retinal. By isolation of the expressed opsins in a stable form, kinetic studies of retinal binding to the opsins in vitro have been carried out by using defined phospholipid–detergent mixtures. The results show wide variation in the rates of 11-cis-retinal binding. Thus, the in vitro reconstitution procedure serves as a probe of the retinal-binding pocket in the opsins. Further, a method is described for purification and characterization of the rhodopsin mutants after retinal binding to the opsins in vitro.

The current biodiversity extinction event: Scenarios for mitigation and recovery

The current massive degradation of habitat and extinction of species is taking place on a catastrophically short timescale, and their effects will fundamentally reset the future evolution of the planet's biota. The fossil record suggests that recovery of global ecosystems has required millions or even tens of millions of years. Thus, intervention by humans, the very agents of the current environmental crisis, is required for any possibility of short-term recovery or maintenance of the biota. Many current recovery efforts have deficiencies, including insufficient information on the diversity and distribution of species, ecological processes, and magnitude and interaction of threats to biodiversity (pollution, overharvesting, climate change, disruption of biogeochemical cycles, introduced or invasive species, habitat loss and fragmentation through land use, disruption of community structure in habitats, and others). A much greater and more urgently applied investment to address these deficiencies is obviously warranted. Conservation and restoration in human-dominated ecosystems must strengthen connections between human activities, such as agricultural or harvesting practices, and relevant research generated in the biological, earth, and atmospheric sciences. Certain threats to biodiversity require intensive international cooperation and input from the scientific community to mitigate their harmful effects, including climate change and alteration of global biogeochemical cycles. In a world already transformed by human activity, the connection between humans and the ecosystems they depend on must frame any strategy for the recovery of the biota.

Structure and function of pectic enzymes: Virulence factors of plant pathogens

The structure and function of Erwinia chrysanthemi pectate lysase C, a plant virulence factor, is reviewed to illustrate one mechanism of pathogenesis at the molecular level. Current investigative topics are discussed in this paper.

Galaxies and large scale structure at high redshifts

It is now straightforward to assemble large samples of very high redshift (z ∼ 3) field galaxies selected by their pronounced spectral discontinuity at the rest frame Lyman limit of hydrogen (at 912 Å). This makes possible both statistical analyses of the properties of the galaxies and the first direct glimpse of the progression of the growth of their large-scale distribution at such an early epoch. Here I present a summary of the progress made in these areas to date and some preliminary results of and future plans for a targeted redshift survey at z = 2.7–3.4. Also discussed is how the same discovery method may be used to obtain a “census” of star formation in the high redshift Universe, and the current implications for the history of galaxy formation as a function of cosmic epoch.

Structure and inhibition of plasmepsin II, a hemoglobin-degrading enzyme from Plasmodium falciparum.

Plasmodium falciparum is the major causative agent of malaria, a disease of worldwide importance. Resistance to current drugs such as chloroquine and mefloquine is spreading at an alarming rate, and our antimalarial armamentarium is almost depleted. The malarial parasite encodes two homologous aspartic proteases, plasmepsins I and II, which are essential components of its hemoglobin-degradation pathway and are novel targets for antimalarial drug development. We have determined the crystal structure of recombinant plasmepsin II complexed with pepstatin A. This represents the first reported crystal structure of a protein from P. falciparum. The crystals contain molecules in two different conformations, revealing a remarkable degree of interdomain flexibility of the enzyme. The structure was used to design a series of selective low molecular weight compounds that inhibit both plasmepsin II and the growth of P. falciparum in culture.

Mapping the mouse genome: current status and future prospects.

The mouse is the best model system for the study of mammalian genetics and physiology. Because of the feasibility and importance of studying genetic crosses, the mouse genetic map has received tremendous attention in recent years. It currently contains over 14,000 genetically mapped markers, including 700 mutant loci, 3500 genes, and 6500 simple sequence length polymorphisms (SSLPs). The mutant loci and genes allow insights and correlations concerning physiology and development. The SSLPs provide highly polymorphic anchor points that allow inheritance to be traced in any cross and provide a scaffold for assembling physical maps. Adequate physical mapping resources--notably large-insert yeast artificial chromosome (YAC) libraries--are available to support positional cloning projects based on the genetic map, but a comprehensive physical map is still a few years away. Large-scale sequencing efforts have not yet begun in mouse, but comparative sequence analysis between mouse and human is likely to provide tremendous information about gene structure and regulation.

Crystal structure of Pseudomonas aeruginosa catabolic ornithine transcarbamoylase at 3.0-A resolution: a different oligomeric organization in the transcarbamoylase family.

The crystal structure of the Glu-105-->Gly mutant of catabolic ornithine transcarbamoylase (OTCase; carbamoyl phosphate + L-ornithine = orthophosphate + L-citrulline, EC 2.1.3.3) from Pseudomonas aeruginosa has been determined at 3.0-A resolution. This mutant is blocked in the active R (relaxed) state. The structure was solved by the molecular replacement method, starting from a crude molecular model built from a trimer of the catalytic subunit of another transcarbamoylase, the extensively studied aspartate transcarbamoylase (ATCase) from Escherichia coli. This model was used to generate initial low-resolution phases at 8-A resolution, which were extended to 3-A by noncrystallographic symmetry averaging. Four phase extensions were required to obtain an electron density map of very high quality from which the final model was built. The structure, including 4020 residues, has been refined to 3-A, and the current crystallographic R value is 0.216. No solvent molecules have been added to the model. The catabolic OTCase is a dodecamer composed of four trimers organized in a tetrahedral manner. Each monomer is composed of two domains. The carbamoyl phosphate binding domain shows a strong structural homology with the equivalent ATCase part. In contrast, the other domain, mainly implicated in the binding of the second substrate (ornithine for OTCase and aspartate for ATCase) is poorly conserved. The quaternary structures of these two allosteric transcarbamoylases are quite divergent: the E. coli ATCase has pseudo-32 point-group symmetry, with six catalytic and six regulatory chains; the catabolic OTCase has 23 point-group symmetry and only catalytic chains. However, both enzymes display homotropic and heterotropic cooperativity.