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Journal Issue - Volume 19 Issue 6 (June 2010)

  • In This Issue

  • Published in Wiley Interscience on May 24, 2010
  • DOI: 10.1002/pro.413 (p )

Abstract We present a method with the potential to generate a library of coil segments from first principles. Proteins are built from α‐helices and/or β‐strands interconnected by these coil segments. Here, we investigate the conformational determinants of short coil segments, with particular emphasis on chain turns. Toward this goal, we extracted a comprehensive set of two‐, three‐, and four‐residue turns from X‐ray–elucidated...

Abstract Tetrapyrroles like hemes, chlorophylls, and cobalamin are complex macrocycles which play essential roles in almost all living organisms. Heme serves as prosthetic group of many proteins involved in fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen. Further, enzymes such as catalases, peroxidases, or cytochromes P450 rely on heme as essential cofactors. Heme is...

Abstract The Lac repressor has been used as a tool to understand protein–DNA recognition for many years. Recent experiments have demonstrated the ability of the Lac repressor to control gene expression in various eukaryotic systems, making the quest for an arsenal of protein–DNA binding partners desirable for potential therapeutic applications. Here, we present the results of the most exhaustive screen of Lac repressor‐DNA binding...

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Abstract Protein export mediated by the general secretory Sec system in Escherichia coli proceeds by a dynamic transfer of a precursor polypeptide from the chaperone SecB to the SecA ATPase motor of the translocon and subsequently into and through the channel of the membrane‐embedded SecYEG heterotrimer. The complex between SecA and SecB is stabilized by several separate sites of contact. Here we have demonstrated directly an interaction...

Abstract Calcium binding in proteins exhibits a wide range of polygonal geometries that relate directly to an equally diverse set of biological functions. The binding process stabilizes protein structures and typically results in local conformational change and/or global restructuring of the backbone. Previously, we established the MUG program, which utilized multiple geometries in the Ca2+‐binding pockets of holoproteins to...

Abstract This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation‐exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size‐exclusion chromatography in separating complex protein mixtures, offering an...

Abstract It was revealed from the crystal structure analysis of S‐ovalbumin (S‐OVA) formed by alkaline treatment that Ser164, Ser236, and Ser320 take the D‐amino acid residue configuration (Yamasaki et al., J Biol Chem 2003; 278:35524–35530). To address the implications of a D‐configuration for these Ser residues in S‐OVA formation, three mutant OVAs (S164A, S236A, and S320A) were generated to compare their thermostabilities before and after alkaline...

Abstract The regulatory subunits of cAMP‐dependent protein kinase (PKA) are the major receptors of cAMP in most eukaryotic cells. As the cyclic nucleotide binding (CNB) domains release cAMP and bind to the catalytic subunit of PKA, they undergo a major conformational change. The change is mediated by the B/C helix in CNB‐A, which extends into one long helix that now separates the two CNB domains and docks onto the surface of the...

Abstract TIS11d is a member of the CCCH‐type family of tandem zinc finger (TZF) proteins; the TZF domain of TIS11d (residues 151–220) is sufficient to bind and destabilize its target mRNAs with high specificity. In this study, the TZF domain of TIS11d is simulated in an aqueous environment in both the free and RNA‐bound states. Multiple nanosecond timescale molecular dynamics trajectories of TIS11d wild‐type and E157R/E195K mutant...

Abstract Fragment complementation has been used to investigate the role of chain connectivity in the catalytic reaction of phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa, a human pathogen. A heterodimer of PMM/PGM, created from fragments corresponding to its first three and fourth domains, was constructed and enzyme activity reconstituted. NMR spectra demonstrate that the fragment corresponding to the fourth...

Abstract A self‐cleaving elastin‐like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP α subunit was tagged with a self‐cleaving ELP‐intein tag and coexpressed with the β, β′, and ω subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove...

Abstract Cren7 is a crenarchaeal conserved chromatin protein discovered recently. To explore the mechanism of the DNA packaging in Crenarchaeota, the crystal structure of Cren7–GCGATCGC complex has been determined and refined at 1.6 Å resolution. Cren7 kinks the dsDNA sharply similar to Sul7d, another chromatin protein existing only in Sulfolobales, which reveals that the “bending and unwinding” compacting mechanism is conserved in...

Abstract The σE‐dependent stress response in bacterial cells is initiated by the DegS‐ and RseP‐regulated intramembrane proteolysis of a membrane‐spanning antisigma factor, RseA. RseB binds to RseA and inhibits its sequential cleavage, thereby functioning as a negative modulator of this response. In the crystal structure of the periplasmic domain of RseA bound to RseB, the DegS cleavage site of RseA is unstructured, however, its P1...

Abstract Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N‐linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence‐activated cell sorting (FACS) and site‐specific gene...

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