The Bacteriophage lambdaDNA packaging enzyme: Identification of four structural domains of the gpNu1 subunit using limited proteolysis

Lambda DNA terminase, the enzyme that cleaves virion-length chromosomes from multigenomic concatemers and packages them into the bacteriophage head, is composed of two subunits, gpNu1 and gpA. Direct determination of the structure of gpNu1, the smaller subunit, has not been possible because of its i...

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Autores principales:	ARAYA,PAMELA, ROSEMBLATT,MARIO, VALENZUELA,PABLO, MURIALDO,HELIOS
Lenguaje:	English
Publicado:	Sociedad de Biología de Chile 2001
Materias:	DNA packaging terminase gpNu1 structural domains limited proteolysis
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602001000300008
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spelling	oai:scielo:S0716-976020010003000082002-06-25The Bacteriophage lambdaDNA packaging enzyme: Identification of four structural domains of the gpNu1 subunit using limited proteolysisARAYA,PAMELAROSEMBLATT,MARIOVALENZUELA,PABLOMURIALDO,HELIOS DNA packaging terminase gpNu1 structural domains limited proteolysis Lambda DNA terminase, the enzyme that cleaves virion-length chromosomes from multigenomic concatemers and packages them into the bacteriophage head, is composed of two subunits, gpNu1 and gpA. Direct determination of the structure of gpNu1, the smaller subunit, has not been possible because of its insolubility in aqueous solutions. Therefore, to identify smaller and potentially water-soluble domains of gpNu1, we analyzed the nature of the products obtained by limited digestion of the protein with several proteases. The gpNu1 subunit was obtained from E.coli cells transfected with the plasmid pH6-Nu1 that overproduces the protein. Incubation of gpNu1 solubized in 2.5 M guanidinium chloride with chymotrypsin resulted in the formation of at least eight discrete protein bands, while treatment with endoproteinase glu-C and bromelain yielded three and one major bands, respectively. The peptides generated by digestion with the various proteases were separated by two-dimensional gel electrophoresis and transferred to Immobilon membranes. Amino acid sequencing of the peptides allowed for the precise assignment of their N-terminal amino acid, while their estimated molecular weights permitted the identification of their C-terminal ends. The results reveal that in the presence of 2.5 M guanidinium chloride, gpNu1 is partially folded in at least four distinct structural domains that correspond to functional domains as determined by previously reported genetic experiments. This information is key to design new plasmids to overproduce these domains for further structural analysis.info:eu-repo/semantics/openAccessSociedad de Biología de ChileBiological Research v.34 n.3-4 20012001-01-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602001000300008en10.4067/S0716-97602001000300008
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	DNA packaging terminase gpNu1 structural domains limited proteolysis
spellingShingle	DNA packaging terminase gpNu1 structural domains limited proteolysis ARAYA,PAMELA ROSEMBLATT,MARIO VALENZUELA,PABLO MURIALDO,HELIOS The Bacteriophage lambdaDNA packaging enzyme: Identification of four structural domains of the gpNu1 subunit using limited proteolysis
description	Lambda DNA terminase, the enzyme that cleaves virion-length chromosomes from multigenomic concatemers and packages them into the bacteriophage head, is composed of two subunits, gpNu1 and gpA. Direct determination of the structure of gpNu1, the smaller subunit, has not been possible because of its insolubility in aqueous solutions. Therefore, to identify smaller and potentially water-soluble domains of gpNu1, we analyzed the nature of the products obtained by limited digestion of the protein with several proteases. The gpNu1 subunit was obtained from E.coli cells transfected with the plasmid pH6-Nu1 that overproduces the protein. Incubation of gpNu1 solubized in 2.5 M guanidinium chloride with chymotrypsin resulted in the formation of at least eight discrete protein bands, while treatment with endoproteinase glu-C and bromelain yielded three and one major bands, respectively. The peptides generated by digestion with the various proteases were separated by two-dimensional gel electrophoresis and transferred to Immobilon membranes. Amino acid sequencing of the peptides allowed for the precise assignment of their N-terminal amino acid, while their estimated molecular weights permitted the identification of their C-terminal ends. The results reveal that in the presence of 2.5 M guanidinium chloride, gpNu1 is partially folded in at least four distinct structural domains that correspond to functional domains as determined by previously reported genetic experiments. This information is key to design new plasmids to overproduce these domains for further structural analysis.
author	ARAYA,PAMELA ROSEMBLATT,MARIO VALENZUELA,PABLO MURIALDO,HELIOS
author_facet	ARAYA,PAMELA ROSEMBLATT,MARIO VALENZUELA,PABLO MURIALDO,HELIOS
author_sort	ARAYA,PAMELA
title	The Bacteriophage lambdaDNA packaging enzyme: Identification of four structural domains of the gpNu1 subunit using limited proteolysis
title_short	The Bacteriophage lambdaDNA packaging enzyme: Identification of four structural domains of the gpNu1 subunit using limited proteolysis
title_full	The Bacteriophage lambdaDNA packaging enzyme: Identification of four structural domains of the gpNu1 subunit using limited proteolysis
title_fullStr	The Bacteriophage lambdaDNA packaging enzyme: Identification of four structural domains of the gpNu1 subunit using limited proteolysis
title_full_unstemmed	The Bacteriophage lambdaDNA packaging enzyme: Identification of four structural domains of the gpNu1 subunit using limited proteolysis
title_sort	bacteriophage lambdadna packaging enzyme: identification of four structural domains of the gpnu1 subunit using limited proteolysis
publisher	Sociedad de Biología de Chile
publishDate	2001
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602001000300008
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The Bacteriophage lambdaDNA packaging enzyme: Identification of four structural domains of the gpNu1 subunit using limited proteolysis

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