title: Rabbit skeletal muscle alpha alpha-tropomyosin expressed in baculovirus-infected insect cells possesses the authentic N-terminus structure and functions.
abstract: When expressed in E. coli, skeletal muscle alpha-tropomyosin has an unacetylated N-terminus. Unacetylated alpha-tropomyosin lacks important functions; this is non-polymerizable and has a low affinity to actin. In the present work, in order to obtain fully functional recombinant alpha-tropomyosin, rabbit skeletal muscle alpha-tropomyosin (alpha-tropomyosin BV) has been expressed in baculovirus-infected insect cells. alpha-TropomyosinBV was not distinguishable from the authentic tropomyosin, not only in functional properties but also in blocked N-terminus. To know the N-terminus structure of alpha-tropomyosinBV, the N-terminal segment six amino acids long, MDAIKK, has been specifically and efficiently removed from alpha-tropomyosinBV by use of an immobilized proteolytic enzyme system based on E. coli cell bodies which carry the ompT gene product, a proteolytic enzyme localized on the outer cell wall of E. coli. The structure of recombinant alpha-tropomyosinBV was shown to be identical to the authentic protein by electrospray mass spectrometry and protein sequencing analysis. Additionally, electrospray mass spectrometry indicated a single phosphorylation not only in alpha- but also beta-tropomyosin chains in the rabbit skeletal muscle. The differentiated susceptibilities of potential ompT cleavage sites are indicative of a non-coiled-coil conformation of the N-terminus of alpha-tropomyosin.
authors.affiliation: European Molecular Biology Laboratory, Hamburg Outstation at DESY, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Kluwe
authors.forename: K
authors.initials: K
authors.lastname: Maeda
authors.forename: A
authors.initials: A
authors.lastname: Miegel
authors.forename: S
authors.initials: S
authors.lastname: Fujita-Becker
authors.forename: Y
authors.initials: Y
authors.lastname: Maéda
authors.forename: G
authors.initials: G
authors.lastname: Talbo
authors.forename: T
authors.initials: T
authors.lastname: Houthaeve
authors.forename: R
authors.initials: R
authors.lastname: Kellner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D018411:Spodoptera; D013329:Structure-Activity Relationship; D014335:Tropomyosin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D014335:Tropomyosin; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease