cDNA was prepared from induced leukocyte poly(A) RNA and cloned in Escherichia coli. Interferon (... more cDNA was prepared from induced leukocyte poly(A) RNA and cloned in Escherichia coli. Interferon (IFN)-alpha cDNA clones were isolated by subculture cloning using a translation hybridization assay. Definitive identification of the clones was based on the production of an interferon-like protein by the transformed bacteria. Different IFN-alpha cDNAs, with characteristic target cell specificities, were identified. The cloned cDNAs typically encode a mature polypeptide of 166 (or, in the case of IFN-alpha 2, 165) amino acids and a signal sequence of 23 amino acids.
the interaction of membrane-bound phosphoinositides with three clinically significant human inos... more the interaction of membrane-bound phosphoinositides with three clinically significant human inositol polyphosphate 5-phosphatases (I5Ps). A comparison to the structures determined with soluble substrates revealed differences in the binding mode and suggested how the I5Ps and apurinic endonuclease (APE1) activities evolved from the same metal-binding active center. A
Bioprocess, Bioseparation, and Cell Technology, 2009
Page 1. PROTEIN AGGREGATION AND PRECIPITATION, MEASUREMENT AND CONTROL CATHERINE H. SCHEIN Struct... more Page 1. PROTEIN AGGREGATION AND PRECIPITATION, MEASUREMENT AND CONTROL CATHERINE H. SCHEIN Structural Biology, University of Texas Medical Branch, Galveston, Texas INTRODUCTION Proteins in solution exist in a precarious equilibrium. ...
Apurinic/apyrimidinic endonuclease 1 (APE1), a central enzyme in the base excision repair pathway... more Apurinic/apyrimidinic endonuclease 1 (APE1), a central enzyme in the base excision repair pathway, cleaves damaged DNA in Mg(2+) dependent reaction. Despite characterization of nine X-ray crystallographic structures of human APE1, in some cases, bound to various metal ions and substrate/product, the position of the metal ion and its stoichiometry for the cleavage reaction are still being debated. While a mutation of the active site E96Q was proposed to eliminate Mg(2+) binding at the "A" site, we show experimentally that this mutant still requires Mg(2+) at concentration similar to that for the wild type enzyme to cleave the AP site in DNA. Molecular dynamics simulations of the wild type APE1, E96Q and a double missense mutant E96Q + D210N indicate that Mg(2+) placed at the A-site destabilizes the bound AP site-containing DNA. In these simulations, the H-bond chain D238-H309-AP site oxygen is broken and the substrate DNA is shifted away from its crystal structure position (1DE9). In contrast, simulations with the Mg(2+) at site B or A+B sites leave the substrate DNA at the position shown in the crystal structure (1DE9). Taken together our MD simulations and biochemical analysis suggests that Mg(2+) binding at the B site is involved in the reaction mechanism associated with endonuclease function of APE1.
In this issue of Structure, Trésaugues and colleagues determined the interaction of membrane-boun... more In this issue of Structure, Trésaugues and colleagues determined the interaction of membrane-bound phosphoinositides with three clinically significant human inositol polyphosphate 5-phosphatases (I5Ps). A comparison to the structures determined with soluble substrates revealed differences in the binding mode and suggested how the I5Ps and apurinic endonuclease (APE1) activities evolved from the same metal-binding active center.
cDNA was prepared from induced leukocyte poly(A) RNA and cloned in Escherichia coli. Interferon (... more cDNA was prepared from induced leukocyte poly(A) RNA and cloned in Escherichia coli. Interferon (IFN)-alpha cDNA clones were isolated by subculture cloning using a translation hybridization assay. Definitive identification of the clones was based on the production of an interferon-like protein by the transformed bacteria. Different IFN-alpha cDNAs, with characteristic target cell specificities, were identified. The cloned cDNAs typically encode a mature polypeptide of 166 (or, in the case of IFN-alpha 2, 165) amino acids and a signal sequence of 23 amino acids.
the interaction of membrane-bound phosphoinositides with three clinically significant human inos... more the interaction of membrane-bound phosphoinositides with three clinically significant human inositol polyphosphate 5-phosphatases (I5Ps). A comparison to the structures determined with soluble substrates revealed differences in the binding mode and suggested how the I5Ps and apurinic endonuclease (APE1) activities evolved from the same metal-binding active center. A
Bioprocess, Bioseparation, and Cell Technology, 2009
Page 1. PROTEIN AGGREGATION AND PRECIPITATION, MEASUREMENT AND CONTROL CATHERINE H. SCHEIN Struct... more Page 1. PROTEIN AGGREGATION AND PRECIPITATION, MEASUREMENT AND CONTROL CATHERINE H. SCHEIN Structural Biology, University of Texas Medical Branch, Galveston, Texas INTRODUCTION Proteins in solution exist in a precarious equilibrium. ...
Apurinic/apyrimidinic endonuclease 1 (APE1), a central enzyme in the base excision repair pathway... more Apurinic/apyrimidinic endonuclease 1 (APE1), a central enzyme in the base excision repair pathway, cleaves damaged DNA in Mg(2+) dependent reaction. Despite characterization of nine X-ray crystallographic structures of human APE1, in some cases, bound to various metal ions and substrate/product, the position of the metal ion and its stoichiometry for the cleavage reaction are still being debated. While a mutation of the active site E96Q was proposed to eliminate Mg(2+) binding at the "A" site, we show experimentally that this mutant still requires Mg(2+) at concentration similar to that for the wild type enzyme to cleave the AP site in DNA. Molecular dynamics simulations of the wild type APE1, E96Q and a double missense mutant E96Q + D210N indicate that Mg(2+) placed at the A-site destabilizes the bound AP site-containing DNA. In these simulations, the H-bond chain D238-H309-AP site oxygen is broken and the substrate DNA is shifted away from its crystal structure position (1DE9). In contrast, simulations with the Mg(2+) at site B or A+B sites leave the substrate DNA at the position shown in the crystal structure (1DE9). Taken together our MD simulations and biochemical analysis suggests that Mg(2+) binding at the B site is involved in the reaction mechanism associated with endonuclease function of APE1.
In this issue of Structure, Trésaugues and colleagues determined the interaction of membrane-boun... more In this issue of Structure, Trésaugues and colleagues determined the interaction of membrane-bound phosphoinositides with three clinically significant human inositol polyphosphate 5-phosphatases (I5Ps). A comparison to the structures determined with soluble substrates revealed differences in the binding mode and suggested how the I5Ps and apurinic endonuclease (APE1) activities evolved from the same metal-binding active center.
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and suggested how the I5Ps and apurinic endonuclease (APE1) activities evolved from the same metal-binding
active center.
A
and suggested how the I5Ps and apurinic endonuclease (APE1) activities evolved from the same metal-binding
active center.
A