Adenylyl cyclase in yeast: antibodies and mutations identify a regulatory domain

The adenylyl cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cAMP from ATP, and two RAS polypeptides, responsible for stimulation of cAMP synthesis by guanine nucleotides. We have obtained rabbit antibodies that recognize the CYR1 protein. Antibodies were raised against synthetic oligopeptides and against a recombinant beta-galactosidase/CYR1 fusion protein. These antibodies have allowed the identification of the CYR1 gene product as a 205 kDa protein. Treatment with trypsin (2 micrograms/ml) reduced the size of the CYR1 protein from 205 to 155 kDa and produced an activated enzyme which no longer responded to guanine nucleotides. This result is consistent with a model in which adenylyl cyclase activity is regulated by an inhibitory domain near the amino-terminus of the CYR1 protein. This model is further supported by the finding that adenylyl cyclase activity is also markedly elevated and unresponsive to guanine nucleotides in mutant yeast strains that express only the carboxy-terminal half of the CYR1 protein. Treatment with high trypsin concentrations (greater than 10 micrograms/ml) caused release of adenylyl cyclase activity from the membrane. Comparison of immunoreactive CYR1 fragments released by trypsin and membrane bound genetically altered proteins suggests that the CYR1 protein is attached to the membrane via a separate trypsin sensitive anchoring protein rather than via a membrane anchoring domain.     

33-kDa C-terminal heparin binding fragment of fibronectin promotes attachment and spreading of hepatocytes

The possibility of interaction of hepatocytes with the heparin binding domain of Fibronectin was examined. Rat hepatocytes adhered to coverslips coated with the 33-kDa heparin binding fragment of the C-terminal region of plasma fibronectin. When different concentrations of the heparin binding fragment were used to coat coverslips and used as substratum, cell attachment showed saturation kinetics. Half the maximum attachment was observed at 30–40 min after seeding of cells. The cells became flat after 2–3 h indicating that they spread on the heparin binding domain as they do on intact fibronectin. Among the different glycosaminoglycans tested, maximum inhibition of attachment was observed for heparin. However it was not possible to completely inhibit attachment even at high concentrations. These results indicate that hepatocytes interact with fibronectin not only through the Arg-Gly-Asp-containing cell binding fragment, but also through the heparin binding domain of fibronectin and, further, that there exist heparin-dependent and heparin-independent mechanisms of interaction of cells with the 33-kDa heparin binding fragment of fibronectin     

Application of three-dimensional molecular hydrophobicity potential to the analysis of spatial organization of membrane domains in proteins: I. Hydrophobic properties of transmembrane segments of Na+, K+-ATPase

A new computer-aided molecular modeling approach based on the concept of three-dimensional (3D) molecular hydrophobicity potential has been developed to calculate the spatial organization of intramembrane domains in proteins. The method has been tested by calculating the arrangement of membrane-spanning segments in the photoreaction center ofRhodopseudomonas viridis and comparing the results obtained with those derived from the X-ray data. We have applied this computational procedure to the analysis of interhelical packing in membrane moiety of Na⁺, K⁺-ATPase. The work consists of three parts. In Part I, 3D distributions of electrostatic and molecular hydrophobicity potentials on the surfaces of transmembrane helical peptides were computed and visualized. The hydrophobic and electrostatic properties of helices are discussed from the point of view of their possible arrangement within the protein molecule. Interlocation of helical segments connected with short extramembrane loops found by means of optimization of their hydrophobic/hydrophilic contacts is considered in Part II. The most probable 3D model of packing of helical peptides in the membrane domain of Na⁺, K⁺-ATPase is discussed in the final part of the work.     

A plastome mutation affects processing of both chloroplast and nuclear DNA-encoded plastid proteins

Summary A bovine tRNA gene cluster has been characterized and the sequences of four tDNAs determined. Two of the tDNAs could encode tRNA^Ser _IGA, one tDNA^Ser _UGA, and the fourth tRNA^Gln _CUG. The three serine tDNAs representing the UCN codon isoacceptor family are almost identical. However, the sequence of the tDNA^Ser _TGA differs from a previously sequenced bovine tDNA^Ser _TGA at 12 positions (ca. 14%). This finding suggests that in the bovine genome, two subfamilies of genes might encode tRNA^Ser _UGA. It also raises the possibility that new genes for a specific UCN isoacceptor might arise from the genes of a different isoacceptor, and could explain previously observed differences between species in the anticodons of coevolving pairs of tRNAs^Ser _UCN. The gene cluster also contains complete and partial copies, and fragments, of the BCS (bovine consensus sequence) SINE (short interspersed nuclear element) family, six examples of which were sequenced. Some of these elements occur in close proximity to two of the serine tDNAs.     

One- and two-dimensional NMR spectral analysis of the consequences of single amino acid replacements in proteins

The traditional approach of using homologous sequences to elucidate the role of specific amino acid residues in protein structure and function becomes more meaningful as the number of differences is minimized, with the limit being alteration of a single residue. For small proteins in solution, NMR spectroscopy offers a means of obtaining detailed information about each residue and its response to a given change in the protein sequence. Extraction of this information has been aided by recent progress in spectrometer technology (higher magnetic fields, more sensitive signal detection, more sophisticated computers) and experimental strategies (new NMR pulse sequences including multiple-quantum and two-dimensional NMR methods). The set of avian ovomucoid third domains, which consists of the third domain proper plus a short leader (connecting peptide) and has a maximum of 56 amino acid residues, offers an attractive system for developing experimental methods for investigating sequence-structure and structure-function relationships in proteins. Our NMR results provide examples of sequence effects on pKa' values, average conformation, and internal motion of amino acid side chains.     

Kinetic characterization of early intermediates in the folding of E. coli tryptophan-synthase beta 2 subunit

This report describes the use of fluorescence energy transfer between an intrinsic energy donor (tryptophan 177) and two chemically added acceptors to study intermediates in the folding of the beta 2 subunit of E. coli tryptophan-synthase. Two early folding steps are thus identified and characterized. One is very rapid (its rate constant at 12 degrees C is 0.02 sec-1) and corresponds to the folding of the N-terminal domain into a structure whose overall features approximate well those of the native domain. The second step is somewhat slower (its rate constant at 12 degrees C is 0.008 sec-1) and involves a conformational rearrangement of the N-terminal domain brought about by the interactions between the N- and C-terminal domains within a monomeric beta chain. This brings to five the number of intermediates which have been identified and ordered on the folding pathway of the dimeric beta 2 subunit.     

Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure,backbone dynamics and protein association

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of¹H-¹⁵N-correlated 2D HSQC,¹⁵N-edited 3D TOCSY-HMQC, and¹⁵N-edited 3D NOESY-HMQC spectra. By analysis of the -proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain.To investigate the internal dynamics of the protein backbone, T₁ and T₂ relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.Abbreviations SH Src homology - GST glutathione-S-transferase - IPTG isopropyl--D-galactopyranoside - DTT dithiothreitol - PMSF phenyl-methyl-sulphonyl-fluoride - TBS 50 mM Tris, 150 mM NaCl, 5 mM DTT, pH 8.0 - MWCO molecular weight cut off - NMR nuclear magnetic resonance - HSQC heteronuclear single-quantum correlation - NOESY nuclear Overhauser effect spectroscopy