Cloning, expression, and protein interaction of human nebulin fragments composed of varying numbers of sequence modules.
Nebulin, a family of giant myofibrillar proteins of 600-900 kDa, contains a large number of highly conserved sequence repeats of 31-38 amino acids. To investigate the significance of this repeat, human skeletal muscle nebulin cDNA fragments encoding two, six, seven, eight, or fifteen repeat modules were expressed in high yield as nonfusion proteins in Escherichia coli with the pET3d plasmid vector. F-actin cosedimentation and solid phase binding assays demonstrated that all nebulin fragments, except the smallest two-module 67-mer, bound to muscle actin with high affinity under physiological ionic conditions. Solid phase binding assays also revealed that a six-module fragment, NB5, binds to myosin and C-terminal protein but fails to bind to tropomyosin, troponin, and tubulin. Furthermore, the binding of NB5 to actin was inhibited by both tropomyosin and troponin. Immunoelectron microscopic localization of NB5 indicated that this N-terminal region fragment is situated near the distal end of thin filaments in the sarcomere. These results indicate that nebulin is a giant protein with an unprecedently large number of actin-binding sites along its length and is anchored at the C terminus to the Z line in the sarcomere. Nebulin may function as a multifunctional template protein that regulates the length of thin filaments and participates in muscle activities by interacting with actin and myosin filaments in the sarcomere of skeletal muscles.
Clayton Foundation Biochemical Institute, Department of Chemistry and Biochemistry, University of Texas 78712.
J P
JP
Jin
K
K
Wang
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012518:Sarcomeres / Q000648:ultrastructure*; D014335:Tropomyosin / Q000378:metabolism; D014336:Troponin / Q000378:metabolism
D000199:Actins; D002352:Carrier Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D010446:Peptide Fragments; D014335:Tropomyosin; D014336:Troponin; C020723:citrate-binding transport protein; C035620:nebulin; D009218:Myosins