Domain structure of tropomodulin: distinct properties of the N-terminal and C-terminal halves.
The structure of tropomodulin, the unique capping protein for the pointed end (the slow-growing end) of an actin filament, was studied. An improved Escherichia coli expression system for chicken E-tropomodulin was established and tropomodulin was prepared, Tmod (N39), in which 15 amino acid residues from the original C-terminus are deleted at the DNA level. This expression and purification system accidentally co-produces an 11-kDa fragment with the original N-terminus (N11). By applying limited proteolysis to Tmod (N39), a 20-kDa C-terminal fragment (C20) was obtained. The limited proteolysis data, as well as the fluorescence spectrometry and CD analyses of Tmod (N39), C20 and N11, revealed that tropomodulin is an alpha-helical protein that consists of two distinct domains. The C-terminal half (20 kDa) is resistant to proteolysis, which suggests that this domain is tightly folded. In contrast, the N-terminal half is susceptible to proteolysis, indicating that in solution this half is likely to be extended or to form a highly flexible structure. Cross-linking experiments with glutaraldehyde indicated that Tmod (N39) and N11 can form complexes with tropomyosin, whereas C20 cannot. This confirms the previous report that the site(s) of interaction with tropomyosin resides in the N-terminal 11-kDa region of tropomodulin.
Kostyukova
A
A
International Institute for Advanced Research, Central Research Laboratories, Matsushita Electric Industrial Co., Ltd. Kyoto, Japan.
Maeda
K
K
Yamauchi
E
E
Krieger
I
I
Maéda
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002645:Chickens; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D015870:Gene Expression; D005976:Glutaral / Q000378:metabolism; D013058:Mass Spectrometry; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D051348:Tropomodulin; D014335:Tropomyosin / Q000378:metabolism
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D051348:Tropomodulin; D014335:Tropomyosin; D010450:Endopeptidases; D005976:Glutaral