Expression of human beta-myosin heavy chain fragments in Escherichia coli; localization of actin interfaces on cardiac myosin.
A cDNA clone coding for an internal fragment of slow-cardiac beta-myosin heavy chain was isolated from a lambda gt10 human skeletal muscle library. Six overlapping cDNA subclones, which span myosin heavy chain subregions and presumably interact with actin, were derived from this clone, fused to a beta-galactosidase vector and expressed in Escherichia coli. Three of the subclones were obtained by PCR (polymerase chain reaction) which enables gene or cDNA fragments to be amplified independently of preexisting restriction sites. Initially, various experiments were carried out using a long MHC (myosin heavy chain) fusion protein containing the 50 kDa-20 kDa connecting region, the whole 20 kDa region and the short subfragment 2 region. This MHC fusion protein was chemically or proteolytically cleaved in the same conditions as the native myosin molecule. Whole and truncated forms of the MHC fusion protein were separated on polyacrylamide gels, electroblotted on nitrocellulose sheets and renatured. They were then assayed in overlay experiments with F-actin and/or myosin light chains in solution. Specific antibodies were used to detect interactions between heavy chain fragments and F-actin or light chains. We thus observed that one long heavy chain fragment synthesized by E. coli behaved like proteolytic or chemical MHC preparations made from native myosin molecules. Two chymotryptic fragments of the MHC fusion protein, which are soluble at low ionic strength, cosedimented with F-actin in solution. Our results demonstrate that, in actin overlay experiments with whole fusion proteins, interactions seem to be due to the heavy chain fragment, not to the bacterial component. All interactions were non ATP-sensitive. We further investigated the possible participation of the six recombinant MHC fragments in contributing to the actomyosin interfaces on the 50 kDa-20 kDa regions of the human cardiac beta-MHC. The present procedure, which enables the synthesis of any MHC fragment independent of any protease site, is a powerful new tool for studying structure-function relationships within the myosin molecule family.
Eldin
P
P
Institut National de la Santé et de la Recherche Médicale, INSERM U300, Faculté de Pharmacie, Montpellier, France.
Le Cunff
M
M
Diederich
K W
KW
Jaenicke
T
T
Cornillon
B
B
Mornet
D
D
Vosberg
H P
HP
Léger
J J
JJ
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D004247:DNA; D009218:Myosins