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Journal Issue - Volume 14 Issue 4 (April 2005)

Abstract The chemical shift of the 129Xe NMR signal has been shown to be extremely sensitive to the local environment around the atom and has been used to follow processes such as ligand binding by bacterial periplasmic binding proteins. Here we show that the 129Xe shift can sense more subtle changes: magnesium binding, BeF3− activation, and peptide binding by the Escherichia coli chemotaxis Y protein. 1H‐15N correlation spectroscopy and X‐ray...

Abstract The M2 protein from influenza A virus is a 97‐amino‐acid protein with a single transmembrane helix that forms proton‐selective channels essential to virus function. The hydrophobic transmembrane domain of the M2 protein (M2TM) contains a sequence motif that mediates the formation of functional tetramers in membrane environments. A variety of structural models have previously been proposed which differ in the degree of helix...

Abstract The protein α‐Synuclein (aS) is a synaptic vesicle‐associated regulator of synaptic strength and dopamine homeostasis with a pathological role in Parkinson's disease. The normal function of aS depends on a membrane‐associated conformation that is adopted upon binding to negatively charged lipid surfaces. Previously we found that the membrane‐binding domain of aS is helical and suggested that it may exhibit an unusual...

Abstract In an attempt to identify leads that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), that might control hyperglycaemia in type 2 diabetes, three new analogs of β‐D‐glucopyranose, 2‐(β‐D‐glucopyranosyl)‐5‐methyl‐1, 3, 4‐oxadiazole, ‐benzothiazole, and ‐benzimidazole were assessed for their potency to inhibit GPb activity. The compounds showed competitive inhibition (with respect...

Abstract Accurate free energy estimation is needed in many predictive tasks. The molecular mechanics/Poisson‐Boltzmann solvent accessible surface area (MM/PBSA) approach has proven to be accurate. However, the correlation between the estimated free energy and the distance (e.g., root mean square deviation [RMSD]) from the most stable conformation is hindered by the strong free energy dependence on minor conformational variations. In...

Abstract Although a 22‐kDa human growth hormone (hGH) is the predicted protein product of the hGH‐N gene, a pleiotropic collection of uncharacterized molecular weight and charge isoforms is also produced. Using chromatography and preparative SDS‐PAGE under reducing conditions we isolated an unusually stable mercaptoethanol‐resistant (MER) 45‐kDa hGH. A 5‐h incubation at 100°C in the presence of 2‐mercaptoethanol was required to convert...

Abstract Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3‐MDa hexadecameric complex, (αβγδ)4. PhK comprises two arched octameric (αβγδ)2 lobes that are oriented back‐to‐back with overall D2 symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually...

Abstract We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH‐groups modified by 5,5′‐dithiobis(2‐nitrobenzoate); (2) O‐R‐type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O‐P‐type dimers of mutant GAPDH from B. stearothermophilus...

Abstract l‐Arginine (l‐Arg) has been widely used as an enhancer of protein renaturation. The mechanism behind its action is still not fully understood. Using hen egg white lysozyme as a model protein, we present data that clearly demonstrate the suppression of the aggregation of denatured protein by l‐Arg. By chemical modification of free cysteines, a series of unfolded lysozyme species were obtained that served as models for...

Abstract Overproduction of proteins from cloned genes using fusion protein expression vectors in Escherichia coli and eukaryotic cells has increased the quantity of protein produced. This approach has been widely used in producing soluble recombinant proteins for structural and functional analysis. One major disadvantage, however, of applying this approach for clinical or bioindustrial uses is that proteolytic removal of the fusion carrier is...

Abstract The infectious agent of prion diseases is identified with PrPSc, a β‐rich, amyloidogenic and partially protease resistant isoform of the cellular glycoprotein, PrPC. To understand the process of prion formation in vivo, we and others have studied defined misfolding pathways of recombinant PrP in vitro. The low‐level infectivity of the in vitro misfolded murine PrP amyloid has recently been reported. Here we analyze the in vitro...

Abstract An Escherichia coli plasmid vector for the high‐level expression of hydrophobic membrane proteins is described. The plasmid, pBCL, directs the expression of a target polypeptide fused to the C terminus of a mutant form of the anti‐apoptotic Bcl‐2 family protein, Bcl‐XL, where the hydrophobic C terminus has been deleted, and Met residues have been mutated to Leu to facilitate CNBr cleavage after a single Met inserted at the...

Abstract The native conformation of host‐encoded cellular prion protein (PrPC) is metastable. As a result of a post‐translational event, PrPC can convert to the scrapie form (PrPSc), which emerges as the essential constituent of infectious prions. Despite thorough research, the mechanism underlying this conformational transition remains unknown. However, several studies have highlighted the importance of the N‐terminal region spanning residues...

Abstract Calbindin D28k (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca2+ homeostasis and enzyme regulation. A combination of biochemical methods and mass spectrometry has been used to identify post‐translational modifications of human calbindin. The protein was studied at 37°C or 50°C in the presence or absence of Ca2+. One deamidation site was identified at position 203 (Asn) under all conditions....

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