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Journal Issue - Volume 3 Issue 11 (November 1994)

Abstract Nerve growth factor (NGF), which has a tertiary structure based on a cluster of 3 cystine disulfides and 2 very extended, but distorted β‐hairpins, is the prototype of a larger family of neurotrophins. Prior to the availability of cloning techniques, the mouse submandibular gland was the richest source of NGF and provided sufficient material to enable its biochemical characterization. It binds as a dimer to at least 2...

Abstract The amino acid sequence identity and potential structural similarity between the subunits of bacterial luciferase and the recently determined structure of the luxF molecule are examined. The unique β/α barrel fold found in luxF appears to be conserved in part in the luciferase subunits. From secondary structural predictions of both luciferase subunits, and from structural comparisons between the protein product of the luxF gene, NFP,...

Abstract The Tyr corner is a conformation in which a tyrosine (residue “Y”) near the beginning or end of an antiparallel β‐strand makes an H bond from its side‐chain OH group to the backbone NH and/or CO of residue Y – 3, Y – 4, or Y – 5 in the nearby connection. The most common “classic” case is a Δ4 Tyr corner (more than 40 examples listed), in which the H bond is to residue Y – 4 and the Tyr x1 is near −60°. Y – 2 is almost...

Abstract We report an interesting case of structural similarity between 2 small, nonhomologous proteins, the third domain of ovomucoid (ovomucoid) and the C‐terminal fragment of ribosomal L7/L12 protein (CTF). The region of similarity consists of a 3‐stranded β‐sheet and an α‐helix. This region is highly similar; the corresponding elements of secondary structure share a common topology, and the RMS difference for “equivalent” Cα...

Abstract The folding of the small (56 residues) highly stable B1 immunoglobulin binding domain (GB1) of streptococcal protein G has been investigated by quenched‐flow deuterium‐hydrogen exchange. This system represents a paradigm for the study of protein folding because it exhibits no complicating features superimposed upon the intrinsic properties of the polypeptide chain. Collapse to a semicompact state exhibiting partial order,...

Abstract The enzyme TEM β‐lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM β‐lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0–1.4 M) concentrations of...

Abstract The backbone resonance assignments have been completed for the apo (1H and 15N) and calcium‐loaded (1H, 15N, and 13C) regulatory N‐domain of chicken skeletal troponin‐C (1–90), using multidimensional homonuclear and heteronuclear NMR spectroscopy. The chemical‐shift information, along with detailed NOE analysis and 3JHNHα coupling constants, permitted the determination and quantification of the Ca2+‐induced secondary structural change in the...

Abstract Electrospray ionization mass spectrometry (ESI‐MS) has proven to be a useful tool for examining noncovalent complexes between proteins and a variety of ligands. It has also been used to distinguish between denatured and refolded forms of proteins. Surfactants are frequently employed to enhance solubilization or to modify the tertiary or quaternary structure of proteins, but are usually considered incompatible with mass...

Abstract The objective of this study was to address the question of whether or not urea and guanidine hydrochloride (GdnHCl) give the same estimates of the stability of a particular protein. We previously suspected that the estimates of protein stability from GdnHCl and urea denaturation data might differ depending on the electrostatic interactions stabilizing the proteins. Therefore, 4 coiled‐coil analogs were designed, where the...

Abstract Straight‐chain, non‐natural, nonpolar amino acids norleucine, norvaline, and α‐amino‐n‐butyric acid at various spacings do not interact with themselves to stabilize helix formation in alanine‐based peptides, but do interact with a Tyr spaced i, i + 4 to stabilize alanine helices, similar to the helix‐stabilizing i, i + 4 Tyr‐Leu and Tyr‐Val interactions reported earlier (Padmanabhan S, Baldwin RL, 1994, J Mol Biol 241:706–713). Leu spaced i, i + 4...

Abstract Glu‐50 of aspartate transcarbamoylase from Escherichia coli forms a set of interdomain bridging interactions between the 2 domains of the catalytic chain; these interactions are critical for stabilization of the high‐activity high‐affinity form of the enzyme. The mutant enzyme with an alanine substituted for Glu‐50 (Glu‐50 → Ala) exhibits significantly reduced activity, little cooperativity, and altered regulatory behavior (Newton ...

Abstract Using site‐directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp‐369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000‐fold lower than the value for the wild‐type enzyme. The D369N enzyme...

Abstract Rationally redesigned variants of the 4‐helix‐bundle protein Rop are described. The novel proteins have simplified, repacked, hydrophobic cores and yet reproduce the structure and native‐like physical properties of the wild‐type protein. The repacked proteins have been characterized thermodynamically and their equilibrium and kinetic thermal and chemical unfolding properties are compared with those of wild‐type Rop. The...

Abstract The citric acid cycle enzyme, malate dehydrogenase (MDH), is a dimer of identical subunits. In the crystal structures of 2 prokaryotic and 2 eukaryotic forms, the subunit interface is conformationally homologous. To determine whether or not the quaternary structure of MDH is linked to the catalytic activity, mutant forms of the enzyme from Escherichia coli have been constructed. Utilizing the high‐resolution structure of E. coli MDH,...

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