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Journal Issue - Volume 16 Issue 3 (March 2007)

Abstract Eph receptors and ephrins play important roles in regulating cell migration and positioning during both normal and oncogenic tissue development. Using a surface plasma resonance (SPR) biosensor, we examined the binding kinetics of representative monomeric and dimeric ephrins to their corresponding Eph receptors and correlated the apparent binding affinity with their functional activity in a neuronal growth cone collapse...

Abstract Computational design of proteins with altered ligand specificity is an emerging method for the creation of new biosensing systems. In this work, we investigated the outcome of site‐directed mutagenesis on the Escherichia coli ribose binding protein (RBP), which is frequently used as a design scaffold for computational searches. A ribose biosensor was first constructed whereby an environmentally sensitive fluorescent probe was...

Abstract In solution, Lys48‐linked di‐ubiquitin exists in dynamic equilibrium between closed and open conformations. To understand the effect of interdomain motion in polyubiquitin chains on their ability to bind ligands, we cyclized di‐ubiquitin by cross‐linking the free C terminus of the proximal ubiquitin with the side chain of residue 48 in the distal ubiquitin, using a chemical cross‐linker, 1,6‐Hexane‐bis‐vinylsulfone. Our NMR...

  • Protein fabrication automation

  • J. Colin Cox, Janel Lape, Mahmood A. Sayed, Homme W. Hellinga
  • Published in Wiley Interscience on Jan 02, 2009
  • DOI: 10.1110/ps.062591607 (p 379-390)

Abstract Facile “writing” of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de...

Abstract The last step of the folding reaction of myoglobin is the incorporation of a prosthetic group. In cells, myoglobin is soluble, while heme resides in the mitochondrial membrane. We report here an exhaustive study of the interactions of apomyoglobin with lipid vesicles. We show that apomyoglobin interacts with large unilamellar vesicles under acidic conditions, and that this requires the presence of negatively charged...

Abstract Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen‐III forming protoporphyrinogen‐IX through a monovinyl intermediate, harderoporphyrinogen. Site‐directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue...

Abstract The pro‐peptide of human nerve growth factor (NGF) functions as an intramolecular chaperone during oxidative renaturation of proNGF in vitro and interacts intramolecularly with the mature part of native proNGF. Here, we analyzed the structure formation and stability of the pro‐peptide in the context of proNGF and its intramolecular interaction with the native mature part. Folding and unfolding of the NGF‐coupled...

Abstract Hydroxylations of octane and lauric acid by Cytochrome P450‐BM3 (CYP102A1) wild‐type and three active site mutants—F87A, L188Q/A74G, and F87V/L188Q/A74G—were rationalized using a combination of substrate orientation from docking, substrate binding statistics from molecular dynamics simulations, and barrier energies for hydrogen atom abstraction from quantum mechanical calculations. Wild‐type BM3 typically hydroxylates...

Abstract Glucose‐1‐phosphate uridylyltransferase, also referred to as UDP‐glucose pyrophosphorylase or UGPase, catalyzes the formation of UDP‐glucose from glucose‐1‐phosphate and UTP. Not surprisingly, given the central role of UDP‐glucose in glycogen synthesis and in the production of glycolipids, glycoproteins, and proteoglycans, the enzyme is ubiquitous in nature. Interestingly, however, the prokaryotic and eukaryotic forms of...

Abstract Thr93, Ser94, Thr140, and Ser306 are conserved in all adenylosuccinate lyases (ASL) and are close to other amino acids previously identified by mutagenesis as being in the active site. To test their involvement in the enzyme's function, each of these amino acids was replaced by alanine. All the mutants exhibit circular dichroism spectra which are similar to that of wild‐type enzyme, indicating there is no appreciable change...

Abstract There is a fundamental conflict between two different views of how proteins fold. Kinetic experiments and theoretical calculations are often interpreted in terms of different population fractions folding through different intermediates in independent unrelated pathways (IUP model). However, detailed structural information indicates that all of the protein population folds through a sequence of intermediates predetermined by...

Abstract The circadian input kinase (CikA) is a major element of the pathway that provides environmental information to the circadian clock of the cyanobacterium Synechococcus elongatus. CikA is a polypeptide of 754 residues and has three recognizable domains: GAF, histidine protein kinase, and receiver‐like. This latter domain of CikA lacks the conserved phospho‐accepting aspartyl residue of bona fide receiver domains and is thus a...

Abstract Protein libraries based on natural scaffolds enable the generation of novel molecular tools and potential therapeutics by directed evolution. Here, we report the design and construction of a high complexity library (30 × 1013 sequences) based on the 10th fibronectin type III domain of human fibronectin (10FnIII). We examined the bacterial expression characteristics and stability of this library using a green fluorescent protein...

Abstract Glucosamine‐6‐phosphate synthase channels ammonia over 18 Å from glutamine at the glutaminase site to fructose‐6P at the synthase site. We have modeled the anisotropic displacements of the glutaminase and synthase domains from the two crystallized states, the enzyme in complex with fructose‐6P or in complex with glucose‐6P and a glutamine affinity analog, using TLS (rigid‐body motion in terms of translation, libration, and...

Abstract The basic differences between the 20 natural amino acid residues are due to differences in their side‐chain structures. This characteristic design of protein building blocks implies that side‐chain–side‐chain interactions play an important, even dominant role in 3D‐structural realization of amino acid codes. Here we present the results of a comparative analysis of the contributions of side‐chain–side‐chain (s‐s) and...

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