Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease

This paper describes the use of novel two-dimensional nuclear magnetic resonance (NMR) pulse sequences to provide insight into protein dynamics. The sequences developed permit the measurement of the relaxation properties of individual nuclei in macromolecules, thereby providing a powerful experimental approach to the study of local protein mobility. For isotopically labeled macromolecules, the sequences enable measurements of heteronuclear nuclear Overhauser effects (NOE) and spin-lattice (T1) and spin-spin (T2) 15N or 13C relaxation times with a sensitivity similar to those of many homonuclear 1H experiments. Because T1 values and heteronuclear NOEs are sensitive to high-frequency motions (10(8)-10(12) s-1) while T2 values are also a function of much slower processes, it is possible to explore dynamic events occurring over a large time scale. We have applied these techniques to investigate the backbone dynamics of the protein staphylococcal nuclease (S. Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+ and labeled uniformly with 15N. T1, T2, and NOE values were obtained for over 100 assigned backbone amide nitrogens in the protein. Values of the order parameter (S), characterizing the extent of rapid 1H-15N bond motions, have been determined. These results suggest that there is no correlation between these rapid small amplitude motions and secondary structure for S. Nase. In contrast, 15N line widths suggest a possible correlation between secondary structure and motions on the millisecond time scale. In particular, the loop region between residues 42 and 56 appears to be considerably more flexible on this slow time scale than the rest of the protein.     

Biochemical characterization of the Drosophila dpp protein, a member of the transforming growth factor beta family of growth factors.

The decapentaplegic (dpp) gene of Drosophila melanogaster is required for pattern formation in the embryo and for viability of the epithelial cells in the imaginal disks. The dpp protein product predicted from the DNA sequence is similar to members of a family of growth factors that includes transforming growth factor beta (TGF-beta). We have produced polyclonal antibodies to a recombinant dpp protein made in bacteria and used a metallothionein promoter to express a dpp cDNA in Drosophila S2 cells. Similar to other proteins in the TGF-beta family, the dpp protein produced by the Drosophila cells was proteolytically cleaved, and both portions of the protein were secreted from the cells. The amino-terminal 47-kilodalton (kDa) peptide was found in the medium and in the proteins adhering to the plastic petri dish. The carboxy-terminal peptide, the region with sequence similarity to the active ligand portion of TGF-beta, was found extracellularly as a 30-kDa homodimer. Most of the 30-kDa homodimer was in the S2 cell protein adsorbed onto the surface of the plastic dish. The dpp protein could be released into solution by increased salt concentration and nonionic detergent. Under these conditions, the amino-terminal and carboxy-terminal portions of dpp were not associated in a stable complex.     

Models of proteolysis of oligomeric enzymes and their applications to the trypsinolysis of citrate synthases.

A simple statistical approach was used to generate predictive models of the proteolysis of multisubunit enzymes in order to correlate the loss of enzyme activity with the loss of native subunit. The models were applied to the trypsinolysis of the citrate synthases of pig heart, Bacillus megaterium and Escherichia coli. With the dimeric citrate synthases (pig heart and B. megaterium) trypsinolysis of one of the subunits appears to destroy the activity of the whole enzymic molecule. The hexameric E. coli citrate synthase behaves like a trimer of dimeric units, each of the dimers behaving similarly to the B. megaterium and pig heart enzymes. Palmitoyl-CoA is required for the trypsinolysis of pig heart citrate synthase, and at relatively high concentrations of this compound trypsinolysis of one subunit leaves the other subunit fully active. Palmitoyl-CoA is not required for the trypsinolysis of the other citrate synthases, and high concentrations of this metabolite do not affect the correlation of proteolysis with inactivation of these enzymes.     

A new repetitive protein from Xenopus laevis skin highly homologous to pancreatic spasmolytic polypeptide

A cDNA sequence has been used to derive the precursor structure of a highly repetitive protein in Xenopus laevis skin. From the sequence of a whole family of secretory proteins can be predicted containing a classical hydrophobic signal sequence at the NH2-terminal end of the precursor. The proteins contain four domains with high homology to porcine pancreatic spasmolytic polypeptide. These four cysteine-rich, presumably physiologically active domains are separated in the molecule by a repetitive element, locating two such domains to the NH2 terminus of the precursor protein and the remaining two to the COOH-terminal end. The separating spacer consists of very unusual, precise, threonine and proline-rich repeats containing 9 residues which could be targets for extensive O-glycosylation. Additionally, processing at two pairs of basic residues is suggested to liberate two polypeptides ("spasmolysins") and "spasmolysin-glycoprotein."     

Mechanisms in volume regulation in Ehrlich ascites tumor cells

The Ehrlich ascites tumor cell has been used as a model of an unspecialized mammalian cell, in an attempt to disclose the mechanisms involved in the regulation of cellular water and salt content. In hypotonic medium Ehrlich cells initially swell as nearly perfect osmometers, but subsequently recover their volume within about 10 min with an associated net loss of KCl, amino acids, taurine and cell water. The net loss of KCl takes place mainly via separate, conductive K+ and Cl- transport pathways, and the net loss of taurine through a passive leak pathway. Ca2+ and calmodulin appear to be involved in the activation of the K+ and Cl- channels, as well as the taurine leak pathway. In hypertonic medium Ehrlich cells initially shrink as osmometers, but subsequently recover their volume with an associated net uptake of KCl and water. In this case, the net uptake of KCl is the result of the activation of an electroneutral, Na+- and Cl- -dependent cotransport system with subsequent replacement of cellular Na+ by extracellular K+ via the Na+/K+ pump. In the present review we describe the ion and taurine transporting systems which have been identified in the plasma membrane of the Ehrlich ascites tumor cell. We have emphasized the selectivity of these transport pathways and their activation mechanisms. Finally, we propose a model for the activation of the conductive K+ and Cl- transport pathways in Ehrlich cells which includes Ca2+, leukotrienes, and inositol phosphate as intracellular second messengers.     

A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.

We have generated transgenic mice that express a diverse repertoire of human sequence immunoglobulins. The expression of this repertoire is directed by light and heavy chain minilocus transgenes comprised of human protein coding sequences in an unrearranged, germ-line configuration. In this paper we describe the construction of these miniloci and the composition of the CDR3 repertoire generated by the transgenic mice. The largest transgene discussed is a heavy chain minilocus that includes human mu and gamma 1 coding sequences together with their respective switch regions. It consists of a single 61 kb DNA fragment propagated in a bacterial plasmid vector. Both human heavy chain classes are expressed in animals that carry the transgene. In light chain transgenic animals the unrearranged minilocus sequences recombine to form VJ joints that use all five human J kappa segments, resulting in a diversity of human-like CDR3 regions. Similarly, in heavy chain transgenics the inserted sequences undergo VDJ joining complete with N region addition to generate a human-like VH CDR3 repertoire. All six human JH segments and at least eight of the ten transgene encoded human D segments are expressed. The transgenic animals described in this paper represent a potential source of human sequence antibodies for in vivo therapeutic applications.     

Characterization and localization of cis-diamminedichloro-platinum(II) adducts on a purified oligonucleotide containing the codons 12 and 13 of H-ras proto-oncogene.

The use of substrates containing well defined adducts at precise sites, is required to perform a careful analysis of the toxic and mutagenic potential of a lesion. As a first step in this direction the octamer 5'-d(CCGGCGGT), containing the sequence of the codons 12 d(GGC) and 13 d(GGT) of the human H-ras gene, was reacted with the antitumoral drug cis-diamminedichloroplatinum(II). The platinated products have been purified by HPLC. A first set of experiments, including enzymatic digestions with nuclease P1 followed by alkaline phosphatase and acid-catalysed hydrolysis, allowed us to determine which bases were engaged in the cis-DDP lesions. Our results indicate that only guanine residues were chelated with cisplatin to yield bifunctional adducts. Furthermore, by performing enzymatic digestions with phosphodiesterases, we have located the adducts with respect to the 5' end of the octamer. Among the purified and characterized platinated oligonucleotides, three present a particular interest, since we have shown here that the cis-d(GpG) adduct is precisely situated either at the d(GGC) or at the d(GGT) or at both sites of their sequence.     

Mapping of FMR1, the gene implicated in fragile X-linked mental retardation, on the mouse X chromosome

A genetic map of the Cf-9 to Dmd region of the mouse X chromosome has been established by typing 100 offspring from a Mus musculus x Mus spretus interspecific backcross for the four loci Cf-9, Cdr, Gabra3, and Dmd. The following order and genetic distances in centimorgans were determined: (Cf-9)-2.4 +/- 1.7-(Cdr)-2.0 +/- 1.4-(Gabra3)-4.1 +/- 2.0-(Dmd). Six backcross offspring carrying X chromosomes with recombination events in the Cdr-Dmd region were identified. These recombination events were used to define the position of Fmr-1, the murine homologue of FMR1, which is the gene implicated in the fragile X syndrome in man, and that of DXS296h, the murine homologue of DXS296. Both Fmr-1 and DXS296h were mapped into the same recombination interval as Gabra3 on the mouse X chromosome. These findings provide strong support for the concept that the order of loci lying in the Cf-9 to Gabra3 segment of the X chromosome is highly conserved between human and mouse.