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Journal Issue - Volume 16 Issue 6 (June 2007)

Abstract The four integral δ subunits of the phosphorylase kinase (PhK) complex are identical to calmodulin (CaM) and confer Ca2+ sensitivity to the enzyme, but bind independently of Ca2+. In addition to binding Ca2+, an obligatory activator of PhK's phosphoryltransferase activity, the δ subunits transmit allosteric signals to PhK's remaining α, β, and γ subunits in activating the enzyme. Under mild conditions about 10% of the δ subunits can be exchanged...

Abstract Prp8 is a critical pre‐mRNA splicing factor. Prp8 is proposed to help form and stabilize the spliceosome catalytic core and to be an important regulator of spliceosome activation. Mutations in human Prp8 (hPrp8) cause a severe form of the genetic disorder retinitis pigmentosa, RP13. Understanding the molecular mechanism of Prp8's function in pre‐mRNA splicing and RP13 has been hindered by its large size (over 2000 amino...

Abstract D‐Glucose/D‐Galactose‐binding protein (GGBP) mediates chemotaxis toward and active transport of glucose and galactose in a number of bacterial species. GGBP, like other periplasmic binding proteins, can exist in open (ligand‐free) and closed (ligand‐bound) states. We report a 0.92 Å resolution structure of GGBP from Escherichia coli in the glucose‐bound state and the first structure of an open, unbound form of GGBP (at 1.55 Å...

Abstract The murine molecule dectin‐1 (known as the β‐glucan receptor in humans) is an immune cell surface receptor implicated in the immunological defense against fungal pathogens. Sequence analysis has indicated that the dectin‐1 extracellular domain is a C‐type lectin‐like domain, and functional studies have established that it binds fungal β‐glucans. We report several dectin‐1 crystal structures, including a high‐resolution...

Abstract The human AF‐6, a scaffold protein between cell membrane‐associated proteins and the actin cytoskeleton, plays an important role in special cell–cell junctions and signal transduction. It can be phosphorylated by the protein kinase Bcr, which allows efficient binding of the C terminus of Bcr to the PDZ domain of AF‐6 and consequently enhances the binding affinity of AF‐6 to Ras. Formation of the AF‐6, Bcr, and Ras ternary...

Abstract Plasmid‐encoded bacterial R67 dihydrofolate reductase (DHFR) is a NADPH‐dependent enzyme unrelated to chromosomal DHFR in amino acid sequence and structure. R67 DHFR is insensitive to the bacterial drug trimethoprim in contrast to chromosomal DHFR. The crystal structure of Q67H mutant of R67 DHFR bound to NADP+ has been determined at 1.15 Å resolution. The cofactor assumes an extended conformation with the nicotinamide ring bound ...

Abstract A nitroxide side chain (R1) has been substituted at single sites along a helix–turn–helix motif in T4 lysozyme (residues 114–135). Together with previously published data, the new sites reported complete a continuous scan through the motif. Mutants with R1 at sites 115 and 118 were selected for crystallographic analysis to identify the structural origins of the corresponding two‐component EPR spectra. At 115, R1 is shown to...

Abstract We evaluate the pKa of dihydrofolate (H2F) at the N5 position in three ternary complexes with Escherichia coli dihydrofolate reductase (ecDHFR), namely ecDHFR(NADP+:H2F) in the closed form (1), and the Michaelis complexes ecDHFR(NADPH:H2F) in the closed (2) and occluded (3) forms, by performing free energy perturbation with molecular dynamics simulations (FEP/MD). Our simulations suggest that in the Michaelis complex the pKa is modulated by the...

Abstract A series of explicit‐solvent molecular dynamics simulations of the protein ubiquitin are reported, which investigate the effect of environmental factors (presence of methanol cosolvent in the aqueous solution, neutral or low pH value, room or elevated temperature) on the structure, stability, and dynamics of the protein. The simulations are initiated either from the native structure of the protein or from a model of a...

Abstract Calmodulin (CaM) is an EF‐hand protein composed of two calcium (Ca2+)‐binding EF‐hand motifs in its N‐domain (EF‐1 and EF‐2) and two in its C‐domain (EF‐3 and EF‐4). In this study, we examined the structure, dynamics, and Ca2+‐binding properties of a fragment of CaM containing only EF‐2 and EF‐3 and the intervening linker sequence (CaM2/3). Based on NMR spectroscopic analyses, Ca2+‐free CaM2/3 is predominantly unfolded, but upon...

Abstract Viral methyltransferases are involved in the mRNA capping process, resulting in the transfer of a methyl group from S‐adenosyl‐L‐methionine to capped RNA. Two groups of methyltransferases (MTases) are known: (guanine‐N7)‐methyltransferases (N7MTases), adding a methyl group onto the N7 atom of guanine, and (nucleoside‐2′‐O‐)‐methyltransferases (2′OMTases), adding a methyl group to a ribose hydroxyl. We have expressed and...

Abstract The understanding of the mechanism of enzymatic recovery of NADH is of biological and of considerable biotechnological interest, since the essential, but expensive, cofactor NADH is exhausted in asymmetric hydrogenation processes, but can be recovered by NAD+‐dependent formate dehydrogenase (FDH). Most accepted for this purpose is the FDH from the yeast Candida boidinii (CbFDH), which, having relatively low thermostability and specific...

Abstract The reaction of adenosylcobalamin‐dependent dioldehydrase with 1,2‐propanediol gives rise to a radical intermediate observable by EPR spectroscopy. This reaction requires a monovalent cation such as potassium ion. The radical signal arises from the formation of a radical pair comprised of the Co(II) of cob(II)alamin and a substrate‐related radical generated upon hydrogen abstraction by the 5′‐deoxyadenosyl radical. The...

Abstract Trigger factor (TF) is the first chaperone to interact with nascent chains and facilitate their folding in bacteria. Escherichia coli TF is 432 residues in length and contains three domains with distinct structural and functional properties. The N‐terminal domain of TF is important for ribosome binding, and the M‐domain carries the PPIase activity. However, the function of the C‐terminal domain remains unclear, and the residues or...

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