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Journal Issue - Volume 14 Issue 8 (August 2005)

Abstract Multiprotein complexes in the cell are dynamic entities that are constantly undergoing changes in subunit composition and conformation to carry out their functions. The protein–DNA complex that promotes recombination of the bacteriophage Mu is a prime example of a complex that must undergo specific changes to carry out its function. The Clp/Hsp100 family of AAA+ ATPases plays a critical role in mediating such changes. The...

Abstract The influence of long‐range residue interactions on defining secondary structure in a protein has long been discussed and is often cited as the current limitation to accurate secondary structure prediction. There are several experimental examples where a local sequence alone is not sufficient to determine its secondary structure, but a comprehensive survey on a large data set has not yet been done. Interestingly, some...

Abstract The expression of human vascular adhesion protein‐1 (hVAP‐1) is induced at sites of inflammation where extravasation of lymphocytes from blood to the peripheral tissue occurs. We have solved the X‐ray structure of hVAP‐1, a human copper amine oxidase (CAO), which is distinguished from other CAOs in being membrane‐bound. The dimer structure reveals some intriguing features that may have fundamental roles in the adhesive and...

Abstract Measles virus is a negative‐sense, single‐stranded RNA virus within theMononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. Themeasles virus nucleoprotein consists of a structured N‐terminal domain, and of an intrinsically disordered C‐terminal domain, NTAIL (aa 401–525), which undergoes induced folding in the presence of the C‐terminal domain (XD, aa 459–507) of the viral...

Abstract Pathogenic Yersinia species use a type III secretion (TTS) system to deliver a number of cytotoxic effector proteins directly into the mammalian host cell. To ensure effective translocation, several such effector proteins transiently bind to specific chaperones in the bacterial cytoplasm. Correspondingly, SycT is the chaperone of YopT, a cysteine protease that cleaves the membrane‐anchor of Rho‐GTPases in the host. We have...

Abstract Acid‐sensing ion channels (ASIC) are proton‐gated sodium channels that have been implicated in pain transduction associated with acidosis in inflamed or ischemic tissues. APETx2, a peptide toxin effector of ASIC3, has been purified from an extract of the sea anemone Anthopleura elegantissima. APETx2 is a 42‐amino‐acid peptide cross‐linked by three disulfide bridges. Its three‐dimensional structure, as determined by...

Abstract We describe a generic, GFP‐based pipeline for membrane protein overexpression and purification in Escherichia coli. We exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane‐integral flavocytochrome. The approach is scalable and suitable for high‐throughput applications. The GFP‐based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important...

Abstract The carboxy‐terminal domain of the transcription factor Escherichia coli NusA, NusACTD, interacts with the protein N of bacteriophage λ, λN, and the carboxyl terminus of the E. coli RNA polymerase α subunit, αCTD. We solved the solution structure of the unbound NusACTD with high‐resolution nuclear magnetic resonance (NMR). Additionally, we investigated the binding sites of λN and αCTD on NusACTD using NMR titrations. The solution ...

Abstract Human γD crystallin (HγD‐Crys) is a two domain, β‐sheet eye lens protein that must remain soluble throughout life for lens transparency. Single amino acid substitutions of HγD‐Crys are associated with juvenile‐onset cataracts. Features of the interface between the two domains conserved among γ‐crystallins are a central six‐residue hydrophobic cluster, and two pairs of interacting residues flanking the cluster. In HγD‐Crys...

Abstract The previously identified dendritic cell‐derived ubiquitin‐like protein (DC‐UbP) was implicated in cellular differentiation and apoptosis. Sequence alignment suggested that it contains a ubiquitin‐like (UbL) domain in the C terminus. Here, we present the solution NMR structure and backbone dynamics of the UbL domain of DC‐UbP. The overall structure of the domain is very similar to that of Ub despite low similarity (<30%)...

Abstract Telomerase is a ribonucleoprotein complex responsible for extending the ends of eukaryotic chromosomes. Structural and biophysical studies of this enzyme have been limited by the inability to produce large amounts of recombinant protein. Here we perform a high‐throughput screen to map regions of the Tetrahymena thermophila TERT (Telomerase Reverse Transcriptase) protein that are overexpressed in a soluble form in Escherichia coli using...

Abstract Probing structures and dynamics within biomolecules using ensemble and single‐molecule fluorescence resonance energy transfer requires the conjugation of fluorophores to proteins in a site‐specific and thermodynamically nonperturbative fashion. Using single‐molecule fluorescence‐aided molecular sorting and the chymotrypsin inhibitor 2–subtilisin BPN′ complex as an example, we demonstrate that protein–protein interactions...

Abstract A new screening procedure is described that uses docking calculations to design enhanced agonist peptides that bind to major histocompatibility complex (MHC) class I receptors. The screening process proceeds via single mutations of one amino acid at the positions that directly interact with the MHC receptor. The energetic and structural effects of these mutations have been studied using fragments of the original ligand that...

Abstract It was recently described that the α5 and the α13 helices of human pancreatic glucokinase play a major role in the allosteric regulation of the enzyme. In order to understand the structural importance of these helices, we have performed site‐directed mutagenesis to generate glucokinase derivatives with altered residues. We have analyzed the kinetic parameters of these mutated forms and compared them with wild‐type and...

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