Four methods that infer protein function and linkages have been combined in a single database, Prolinks, which spans 83 organisms and includes 10 million high-confidence links.
A major focus of genome research is to decipher the networks of molecular interactions that underlie cellular function. We describe a computational approach for identifying detailed relationships between proteins on the basis of genomic data. Logic analysis of phylogenetic profiles identifies triplets of proteins whose presence or absence obey certain logic relationships. For example, protein C may be present in a genome only if proteins A and B are both present. The method reveals many previously unidentified higher order relationships. These relationships illustrate the complexities that arise in cellular networks because of branching and alternate pathways, and they also facilitate assignment of cellular functions to uncharacterized proteins.
A novel approach has been developed for the isolation and maturation of human antibodies that replicates key features of the adaptive immune system by coupling in vitro somatic hypermutation (SHM) with mammalian cell display. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID), and can be replicated in non-B cells through expression of recombinant AID. A library of human antibodies, based on germline V-gene segments with recombined human D J regions was used to isolate low-affinity antibodies to human β nerve growth factor (hβNGF). These antibodies, initially naïve to SHM, were subjected to AID-directed SHM in vitro and selected using the same mammalian cell display system, as illustrated by the maturation of one of the antibodies to low pM K D . This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs. affinity maturation | mammalian display
We have measured deuterium/hydrogen fractionation in three histidine-containing proteins, ecHPr, ecHPr mutant S31A, and bsHPr, and in random coil peptides using NMR and mass spectrometry. The amide protons of unstructured peptides exhibit equilibrium enrichment for deuterium, in agreement with previous studies. Enrichment for both protium and deuterium was observed in both HPrs, with fractionation factors ranging from 0.63 to 1.41. Enrichment for protium was seen in alpha-helical secondary structure. 'Strong' HBs previously identified by mutagenesis and thermodynamic measurements are significantly enriched for protium. Sites of protium enrichment are conserved in a structural context across species lines, though ecHPr and bsHPr share only 30% sequence identity, suggesting that strong HBs are conserved and may play an important role in stabilizing the folded state.
The most widespread of all Holocene tephra deposits in the Cook Inlet region of south-central Alaska is a set of deposits from Hayes volcano. Because of their unique phenocryst content—biotite in rare amounts and a high proportion of amphibole to pyroxene—the deposits are readily identifiable at all but the most distant sites where they are very fine grained. Eighteen radiocarbon dates from eight upland sites limit the age of the tephra set to between about 3500 and 3800 yr. The set originated at Hayes volcano in the Tordrillo Mountains 150 km northwest of Anchorage; seven or possibly eight closely succeeding deposits, low-silica dacite in composition, compose two main lobes that extend northeast for 400 km and south for at least 250 km from the vent. We estimate the total tephra volume to be 10 km3; multiple layers imply four to six larger and two or three smaller eruptions. The deposits are a nearly isochronous marker horizon that should be useful in future archeologic, geologic, and palynologic studies in the region.
We describe a method for generating moderate to high-resolution protein structures using limited NMR data combined with the ab initio protein structure prediction method Rosetta. Peptide fragments are selected from proteins of known structure based on sequence similarity and consistency with chemical shift and NOE data. Models are built from these fragments by minimizing an energy function that favors hydrophobic burial, strand pairing, and satisfaction of NOE constraints. Models generated using this procedure with approximately 1 NOE constraint per residue are in some cases closer to the corresponding X-ray structures than the published NMR solution structures. The method requires only the sparse constraints available during initial stages of NMR structure determination, and thus holds promise for increasing the speed with which protein solution structures can be determined.
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