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Journal Issue - Volume 17 Issue 7 (July 2008)

Abstract In order to infect their hosts, many Gram‐negative bacteria translocate agents of infection, called effector proteins, through the type III secretion system (TTSS) into the host cytoplasm. This process is thought to require at least partial unfolding of these agents, raising the question of how an effector protein might unfold to enable its translocation and then refold once it reaches the host cytoplasm. AvrPto is a...

Abstract Evolutionarily conserved triad glutamine amidotransferase (GAT) domains catalyze the cleavage of glutamine to yield ammonia and sequester the ammonia in a tunnel until delivery to a variety of acceptor substrates in synthetase domains of variable structure. Whereas a conserved hydrolytic triad (Cys/His/Glu) is observed in the solved GAT structures, the specificity pocket for glutamine is not apparent, presumably because its...

Abstract During systematic analysis of nonbonded contacts in protein–ligand complexes derived from crystal structures in the Protein Data Bank, Cl–π interactions have been found, not only in the well‐documented serine proteases but also, to a lesser extent, in other proteins. From geometric analysis of such Cl–π interactions in the crystal structures, two distinct geometries were found: the “edge‐on” approach of a Cl atom to a ring...

Abstract Bacterioferritins, also known as cytochrome b1, are oligomeric iron‐storage proteins consisting of 24 identical amino acid chains, which form spherical particles consisting of 24 subunits and exhibiting 432 point‐group symmetry. They contain one haem b molecule at the interface between two subunits and a di‐nuclear metal binding center. The X‐ray structure of bacterioferritin from Mycobacterium smegmatis (Ms‐Bfr) was determined to a...

Abstract Globular proteins adopt complex folds, composed of organized assemblies of α‐helix and β‐sheet together with irregular regions that interconnect these scaffold elements. Here, we seek to parse the irregular regions into their structural constituents and to rationalize their formative energetics. Toward this end, we dissected the Protein Coil Library, a structural database of protein segments that are neither α‐helix nor...

Abstract Adenylosuccinate lyase (ASL) catalyzes two β‐elimination reactions in purine biosynthesis, leading to the question of whether the two substrates occupy the same or different active sites. Kinetic studies of Bacillus subtilis and human ASL with a new substrate analog, adenosine phosphonobutyric acid, 2′(3′), 5′‐diphosphate (APBADP), show that it acts as a competitive inhibitor with respect to either substrate (KI ∼ 0.1 μM), indicating...

Abstract A crystallization chaperone is an auxiliary protein that binds to a target of interest, enhances and modulates crystal packing, and provides high‐quality phasing information. We critically evaluated the effectiveness of a camelid single‐domain antibody (VHH) as a crystallization chaperone. By using a yeast surface display system for VHH, we successfully introduced additional Met residues in the core of the VHH scaffold. We identified a...

Abstract G‐protein‐coupled receptors (GPCRs) must properly insert and fold in the membrane to adopt a stable native structure and become biologically active. The interactions between transmembrane (TM) helices are believed to play a major role in these processes. Previous studies in our group showed that specific interactions between TM helices occur, leading to an increase in helical content, especially in weakly helical TM...

Abstract The left‐handed polyproline II helical structure (PII) is observed to be a dominant conformation in the disordered states of protein and small polypeptide chains, even when no prolines are present in the sequence. Recently, in work by Ferreon and Hilser, the energetics associated with Ala and Gly substitutions at a surface exposed proline site were determined calorimetrically by measuring the binding energetics of Sos ...

Abstract One of the common methods for assessing energy functions of proteins is selection of native or near‐native structures from decoys. This is an efficient but indirect test of the energy functions because decoy structures are typically generated either by sampling procedures or by a separate energy function. As a result, these decoys may not contain the global minimum structure that reflects the true folding accuracy of the...

Abstract The determination of the location and conformation of a natural ligand bound to a protein receptor is often a first step in the rational design of molecules that can modulate receptor function. NMR observables, including NOEs, often provide the basis for these determinations. However, when ligands are carbohydrates, interactions mediated by extensive hydrogen‐bonding networks often reduce or eliminate NOEs between ligand...

Abstract Alanine scanning has been widely employed as a method of identifying side chains that play important roles in protein–protein and protein–peptide interactions. Here we show how an analogous and complementary technique, hydrophile scanning, can provide additional insight on such interactions. Mutation of a wild‐type residue to alanine removes most of the side‐chain atoms, and the effect of this removal is typically...

Abstract Expression of recombinant proteins as fusions with SUMO (small ubiquitin‐related modifier) protein has significantly increased the yield of difficult‐to‐express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native ...

Abstract The proline‐rich designer antibacterial peptide dimer A3‐APO is currently under preclinical development for the treatment of systemic infections caused by antibiotic‐resistant Gram‐negative bacteria. The peptide showed remarkable stability in 25% mouse serum in vitro, exhibiting a half‐life of ∼100 min as documented by reversed‐phase chromatography. Indeed, after a 30‐min incubation period in undiluted mouse serum ex vivo,...

Abstract Protein folding speeds are known to vary over more than eight orders of magnitude. Plaxco, Simons, and Baker (see References) first showed a correlation of folding speed with the topology of the native protein. That and subsequent studies showed, if the native structure of a protein is known, its folding speed can be predicted reasonably well through a correlation with the “localness” of the contacts in the protein. In the...

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