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Journal Issue - Volume 7 Issue 7 (July 1998)

Abstract The structure has been determined at 3.0 Å resolution of a complex of engineered monomeric Cro repressor with a seven‐base pair DNA fragment. Although the sequence of the DNA corresponds to the consensus half‐operator that is recognized by each subunit of the wild‐type Cro dimer, the complex that is formed in the crystals by the isolated monomer appears to correspond to a sequence‐independent mode of association. The...

Abstract We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C‐terminus of the active‐site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8, 000 amino acid combinations. The most active of these 8, 000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli,...

Abstract The conformational properties of an 18 residues peptide spanning the entire sequence, L1KTPA5QFDAD10ELRAA15MKG, of the first helix (A‐helix) of domain 2 of annexin I, were thoroughly investigated. This fragment exhibits several singular features, and in particular, two successive potential capping boxes, T3XXQ6 and D8XXE11. The former corresponds to the native hydrogen bond network stabilizing the α helix N‐terminus in the protein; the latter ...

Abstract Off‐pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self‐association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off‐pathway aggregation of the parallel β‐coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in...

Abstract The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide‐induced conformational changes in GroEL, no details are available about the specific...

Abstract Flavocytochrome b2 or L‐lactate dehydrogenase from yeasts catalyzes the oxidation of L‐lactate at the expense of monoelectronic acceptors such as cytochrome c, its physiological partner. When incubated in the presence of both L‐lactate and a keto acid, the enzyme catalyzes a transhydrogenation reaction wherein only the flavin is involved. During this reaction, the substrate α‐hydrogen is transferred not only to the solvent...

Abstract The stabilities of Bucillus circuluns xylanase and a disulfide‐bridge‐containing mutant (S100C/N148C) were investigated by differential scanning calorimetry (DSC) and thermal inactivation kinetics. The thermal denaturation of both proteins was found to be irreversible, and the apparent transition temperatures showed a considerable dependence upon scanning rate. In the presence of low (nondenaturing) concentrations of urea,...

Abstract The betabellin target structure is a β‐sandwich protein consisting of two 32 residue β‐sheets packed against one another by interaction of their hydrophobic faces. The 32 residue chain of betabellin‐15S (HSLTAKIpkLTFSIAphTYTCAV pkYTAKVSH, where p = DPrO, k = DLYS, and h = DHis) did not fold in water at pH 6.5. Air oxidation of betabellin‐15S provided betabellin‐15D, the 64 residue disulfide bridged two‐chain molecule, which...

Abstract The 1H and 13C NMR spectra of a 14‐residue antifreeze glycopeptide from Antarctic cod (Tetramatornus horchgrevinki) containing two proline residues have been assigned. 13C NMR relaxation experiments indicate motional anisotropy of the peptide, with a tumbling time in water at 5 °C of 3‐4 ns. The relaxation data and lack of long‐range NOEs are consistent with a linear peptide undergoing significant segmental motion. However, extreme...

Abstract The guanidine hydrochloride concentration dependence of the folding and unfolding rate constants of a derivative of α‐lactalbumin, in which the 6‐120 disulfide bond is selectively reduced and S‐carboxymethylated, was measured and compared with that of disulfide‐intact α‐lactalbumin. The concentration dependence of the folding and unfolding rate constants was analyzed on the basis of the two alternative models, the...

Abstract Reverse transcriptase is an essential retroviral enzyme that uses RNA‐ and DNA‐directed DNA polymerase activities as well as an RNaseH activity to synthesize a double‐stranded DNA copy of the single‐stranded RNA genome. In an effort to obtain high‐resolution structural information regarding the polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus...

Abstract This paper describes a simple biomimetic strategy to prepare small cyclic proteins containing multiple disulfide bonds. Our strategy involves intramolecular acyl transfer reactions to assist both the synthesis and fragmentation of these highly constrained cyclic structures in aqueous solution. To illustrate our strategy, we synthesized the naturally occurring circulin B and cyclopsychotride (CPT), both consisting of 31...

Abstract An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N‐acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were...

Abstract The general diffusion porin from Rhodopseudomonas blastica was produced in large amounts in Escherichia coli inclusion bodies and (re)natured to the exact native structure. Here, we report on 13 mutants at the pore eyelet giving rise to new diffusion properties as measured in planar lipid bilayer experiments. The crystal structures of seven of these mutants were established. The effects of charge‐modifying mutations at the pore eyelet...

Abstract We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type I (HTLV‐1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after...

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