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Journal Issue - Volume 5 Issue 6 (June 1996)

Abstract Future research on protein folding must confront two serious dilemmas. (1) It may never be possible to observe at high resolution the very important structures that form in the first few milliseconds of the refolding reaction. (2) The energy functions used to predict structure from sequence will always be approximations of the true energy function. One strategy to resolve both dilemmas is to view protein folding from a...

Abstract It is well established that sequence templates (e.g., PROSITE) and databases are powerful tools for identifying biological function and tertiary structure for an unknown protein sequence. Here we describe a method for automatically deriving 3D templates from the protein structures deposited in the Brookhaven Protein Data Bank. As an example, we describe a template derived for the Ser‐His‐Asp catalytic triad found in the...

Abstract The structures of Escherichia coli soluble inorganic pyrophosphatase (E‐PPase) and Thermus thermophilus soluble inorganic pyrophosphatase (T‐PPase) have been compared to find the basis for the superior thermostability of T‐PPase. Both enzymes are D3 hexamers and crystallize in the same space group with very similar cell dimensions. Two rather small changes occur in the T‐PPase monomer: a systematic removal of Ser residues and insertion...

Abstract The structures of several variants of staphylococcal nuclease with long flexible unnatural amino acid side chains in the hydrophobic core have been determined by X‐ray crystallography. The unnatural amino acids are disulfide moieties between the lone cysteine residue in V23C nuclease and methane, ethane, 1‐n‐propane, 1‐n‐butane, 1‐n‐pentane, and 2‐hydroxyethyl thiols. We have examined changes in the core packing of these mutants. Side...

Abstract Reaction of rat liver glutathione S‐transferase, isozyme 1–1, with 4‐(fluorosulfonyl)benzoic acid (4‐FSB), a xenobiotic substrate analogue, results in a time‐dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with l‐chloro‐2,4‐dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4‐FSB from 0.25 mM to 9...

Abstract A quantitative form of the principle of minimal frustration is used to obtain from a database analysis statistical mechanical energy functions and gap parameters for aligning sequences to three‐dimensional structures. The analysis that partially takes into account correlations in the energy landscape improves upon the previous approximations of Goldstein et al. (1994, 1995) (Goldstein R, Luthey‐Schulten Z, Wolynes P, 1994,...

Abstract We use mass spectrometry in conjunction with hydrogen‐deuterium exchange and NMR to characterize the conformational dynamics of the 62‐residue IgG binding domain of protein L under conditions in which the native state is marginally stable. Mass spectra of protein L after short incubations in D2O reveal the presence of two distinct populations containing different numbers of protected protons. NMR experiments indicate that...

Abstract A new conformational database potential involving dihedral angle relationships in databases of high‐resolution highly refined protein crystal structures is presented as a method for improving the quality of structures generated from NMR data. The rationale for this procedure is based on the observation that uncertainties in the description of the nonbonded contacts present a key limiting factor in the attainable accuracy of...

Abstract The crystal structure of the 252‐residue lumen‐side domain of reduced cytochrome f, a subunit of the proton‐pumping integral cytochrome b6f complex of oxygenic photosynthetic membranes, was determined to a resolution of 1.96 Å from crystals cooled to —35°. The model was refined to an R‐factor of 15.8% with a 0.013‐Å RMS deviation of bond lengths from ideality. Compared to the structure of cytochrome f at 20°, the structure at —35° has a small...

Abstract The nucleotide‐binding component of the high‐affinity ribose transport system of Escherichia coli, RbsA, was overproduced from a T7–7 expression vector, and the protein was purified. Biochemical analyses of the purified protein indicated that the ATP analogues, 5′‐FSBA and 8‐azido ATP, covalently labeled the protein, a reaction that was inhibited by ATP, but not by GTP or CTP. The pure protein exhibited low‐level ATPase activity with a...

Abstract Chimeric genes encoding full‐length copies of rbsA and rbsC connected by segments coding for short bridge peptides were constructed and expressed in Escherichia coli. Surprisingly, the chimeric genes complemented the strain in which rbsA and rbsC were deleted. The chimeric proteins were overproduced, and the products were purified by affinity chromatography. In order to obtain highly purified protein, a poly‐His leader peptide was incorporated so...

Abstract The native state fluorescence and CD spectra of the predominantly β‐sheet cellular retinoic acid‐binding protein I (CRABPI) include contributions from its three tryptophan residues and are influenced by the positions of these residues in the three‐dimensional structure. Using a combination of spectroscopic approaches and single Trp‐mutants of CRABPI, we have deconvoluted these spectra and uncovered several features that...

Abstract Bacillus circulans xylanase (BCX) is a member of the family of low molecular weight endo‐β‐(1,4)‐xylanases. The main‐chain 1H, 13C, and 15N resonances of this 20.4‐kDa enzyme were assigned using heteronuclear NMR experiments recorded on a combination of selectively and uniformly labeled protein samples. Using chemical shift, NOE, J coupling, and amide hydrogen exchange information, 14 β‐strands, arranged in a network of three β‐sheets,...

Abstract The question of parallel (α/β)8‐barrel fold evolution remains unclear, owing mainly to the lack of sequence homology throughout the amino acid sequences of (α/β)8‐barrel enzymes. The “classical” approaches used in the search for homologies among (α/β)8‐barrels (e.g., production of structurally based alignments) have yielded alignments perfect from the structural point of view, but the approaches have been unable to reveal the...

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