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Journal Issue - Volume 4 Issue 6 (June 1995)

Abstract The hepatic bifunctional enzyme, 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (6PF‐2‐K/Fru‐2,6‐P2ase), E.C. 2–7–1–105/E.C. 3–1–3–46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose‐2,6‐bisphosphate (Fru‐2,6‐P2). Fru‐2,6‐P2 is a potent activator of the glycolytic enzyme 6‐phosphofructo‐1‐kinase and an inhibitor of the gluconeogenic enzyme...

Abstract Positive cooperativity, defined as an enhancement of the ligand affinity at one site as a consequence of binding the same type of ligand at another site, is a free energy coupling between binding sites. It can be present both in systems with sites having identical ligand affinities and in systems where the binding sites have different affinities. When the sites have widely different affinities such that they are filled with...

Abstract Calbindin D9k is a small EF‐hand protein that binds two calcium ions with positive cooperativity. The molecular basis of cooperativity for the binding pathway where the first ion binds in the N‐terminal site (I) is investigated by NMR experiments on the half‐saturated state of the N56A mutant, which exhibits sequential yet cooperative binding (Linse S, Chazin WJ, 1995, Protein Sci 4:1038–1044). Analysis of calcium‐induced changes in...

Abstract Cellobiohydrolase I (CBHI) of Trichoderma reesei has two functional domains, a catalytic core domain and a cellulose binding domain (CBD). The structure of the CBD reveals two distinct faces, one of which is flat and the other rough. Several other fungal cellulolytic enzymes have similar two‐domain structures, in which the CBDs show a conserved primary structure. Here we have evaluated the contributions of conserved amino ...

Abstract P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane‐bound proteins, they have resisted structure‐function analysis by means of X‐ray crystallographic methods. Among P450‐catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the...

Abstract The three‐dimensional X‐ray structure of a complex of the potent neuraminidase inhibitor 4‐guanidino‐Neu5Ac2en and influenza virus neuraminidase (Subtype N9) has been obtained utilizing diffraction data to 1.8 Å resolution. The interactions of the inhibitor, solvent water molecules, and the active site residues have been accurately determined. Six water molecules bound in the native structure have been displaced by the...

Abstract A simple biochemical method that combines enzymatic proteolysis and matrix‐assisted laser desorption ionization mass spectrometry was developed to probe the solution structure of DNA‐binding proteins. The method is based on inferring structural information from determinations of protection against enzymatic proteolysis, as governed by solvent accessibility and protein flexibility. This approach was applied to the study of...

Abstract The transmembrane domain of chemoreceptor Trg from Escherichia coli contains four transmembrane segments in its native homodimer, two from each subunit. We had previously used mutational analysis and sulfhydryl cross‐linking between introduced cysteines to obtain data relevant to the three‐dimensional organization of this domain. In the current study we used Fourier analysis to assess these data quantitatively for periodicity along the...

Abstract We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First,...

Abstract SecB, a molecular chaperone involved in protein export in Escherichia coli, displays the remarkable ability to selectively bind many different polypeptide ligands whose only common feature is that of being nonnative. The selectivity is explained in part by a kinetic partitioning between the folding of a polypeptide and its association with SecB. SecB has no affinity for native, stably folded polypeptides but interacts tightly with...

Abstract Theoretical computations (molecular dynamics and combined quantum chemical and molecular mechanical geometry optimizations) have been performed on horse liver alcohol dehydrogenase. The results provide evidence that Glu‐68, a highly conserved residue located 0.47 nm from the catalytic zinc ion, may intermittently coordinate to the zinc ion. Structures with Glu‐68 coordinated to the zinc ion are almost as stable as...

Abstract Equilibrium unfolding of barstar with guanidine hydrochloride (GdnHCl) and urea as denaturants as well as thermal unfolding have been carried out as a function of pH using fluorescence, far‐UV and near‐UV CD, and absorbance as probes. Both GdnHCl‐induced and urea‐induced denaturation studies at pH 7 show that barstar unfolds through a two‐state F ⇌ U mechanism and yields identical values for ΔGU the free energy difference between the...

Abstract We have compared commonly used sequence comparison algorithms, scoring matrices, and gap penalties using a method that identifies statistically significant differences in performance. Search sensitivity with either the Smith‐Waterman algorithm or FASTA is significantly improved by using modern scoring matrices, such as BLOSUM45–55, and optimized gap penalties instead of the conventional PAM250 matrix. More dramatic...

Abstract The helix/coil equilibrium of a peptide in solution can be modulated by a variety of side‐chain interactions that are not incorporated into the standard statistical mechanical models for prediction of peptide helical content. In this report, we describe a recursive formulation of the Lifson‐Roig model that facilitates incorporation of specific pairwise side‐chain interactions as well as nonspecific individual side‐chain...

Abstract By means of Monte Carlo simulation, we investigated the equilibrium between folded and unfolded states of lattice model proteins. The amino acid sequences were designed to have pronounced energy minimum target conformations of different length and shape. For short fully compact (36‐mer) proteins, the all‐or‐none transition from the unfolded state to the native state was observed. This was not always the case for longer...

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