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Journal Issue - Volume 3 Issue 2 (February 1994)

Abstract The crystal structures of the ligand binding domain of a bacterial aspartate receptor suggest a simple mechanism for transmembrane signaling by the dimer of the receptor. On ligand binding, one domain rotates with respect to the other, and this rotational motion is proposed to be transmitted through the membrane to the cytoplasmic domains of the receptor.

Abstract Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B‐pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B‐pentamer complexed with the GM1 pentasaccharide. Each...

Abstract The crystal structure of ternary and binary substrate complexes of the catalytic subunit of CAMP‐dependent protein kinase has been refined at 2.2 and 2.25 Å resolution, respectively. The ternary complex contains ADP and a 20‐residue substrate peptide, whereas the binary complex contains the phosphorylated substrate peptide. These 2 structures were refined to crystallographic R‐factors of 17.5 and 18.1%, respectively. In the...

Abstract The active site of acetylcholinesterase (AChE) from Torpedo californica is located 20 Å from the enzyme surface at the bottom of a narrow gorge. To understand the role of this gorge in the function of AChE, we have studied simulations of its molecular dynamics. When simulations were conducted with pure water filling the gorge, residues in the vicinity of the active site deviated quickly and markedly from the crystal structure. Further...

Abstract We describe a new paradigm for modeling proteins in interactive computer graphics systems — continual maintenance of a physically valid representation, combined with direct user control and visualization. This is achieved by a fast algorithm for energy minimization, capable of real‐time performance on all atoms of a small protein, plus graphically specified user tugs. The modeling system, called Sculpt, rigidly constrains...

Abstract The electrostatic contribution to the free energy of folding was calculated fo 21 salt bridges in 9 r X‐ray crystal structures using a continuum electrostatic approach with the DELPHI computer‐program package. The majority (17) were found to be electrostatically destabilizing; the average free energy change, which is analogous to mutation of salt bridging side chains to hydrophobic isosteres, was calculated to be 3.5...

Abstract The Cys 2‐Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease Fl. This C2S/ClON variant resembles the wild‐type protein in structure and in catalytic activity. Minor structural changes were observed by 2‐dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is...

Abstract Using a functional lactose permease mutant devoid of Cys residues (C‐less permease), each amino acid residue in the hydrophilic N‐terminus and the first putative transmembrane helix was systematically replaced with Cys (from Tyr‐2 to Trp‐33). Twenty‐three of 32 mutants exhibit high lactose accumulation (70‐100% or more of C‐less), and an additional 8 mutants accumulate to lower but highly significant levels, Surprisingly,...

Abstract Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisincatalyzed semisynthesis starting from synthetic RNase 1‐20 peptides and S‐protein (RNase 21‐124). The lysyl residue at position 1 was replaced by alanine, whereas Lys‐7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods...

Abstract The sequence‐specific DNA binding of recombinant p42 and p51 ETSl oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETSl or the phosphorylation of expressed Exon VII in p51 ETSl had an effect on DNA binding activity. The kinetics of sequence‐specific DNA binding was measured using real‐time changes in surface plasmon resonance with BIAcore (registered...

Abstract Proton NMR experiments were carried out on apomyoglobin from sperm whale and horse skeletal muscle. Two small molecules, the paramagnetic relaxation agent 4‐hydroxy‐2,2,6,6‐tetramethylpiperidinyl‐l‐oxy (HyTEMPO) and the fluorescent dye 8‐anilino‐1‐naphthalenesulfonic acid (ANS), were used to alter and simplify the spectrum. Both were shown to bind in the heme pocket by docking onto the hydrophobic residues lining the distal...

Abstract The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose‐specific phosphoenol‐pyruvate‐dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone 1H, 15N, and 13Cα resonances, and the HN(CA)CO and HNCO experiments,...

  • An Ecballium elaterium (EETI‐II)

  • Katherine J. Nielsen, Dianne Alewood, John Andrews, Stephen B.H. Kent, David J. Craik
  • Published in Wiley Interscience on Dec 31, 2008
  • DOI: 10.1002/pro.5560030213 (p 291-302)

Abstract The 3‐dimensional structures of mirror‐image forms of a Leu‐5 variant of the trypsin inhibitor Ecballium elaterium (EETI‐II) have been determined by 1H NMR spectroscopy and simulated annealing calculations incorporating NOE‐derived distance constraints. Spectra were assigned using 2‐dimensional NMR methods at 400 MHz, and internuclear distances were determined from NOESY experiments. Three‐bond spin‐spin couplings between CαH and amide...

Abstract The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48‐kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (Gt) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44‐kDa form of arrestin, called p44, from fresh...

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