JKMRS Volume 19, No 2, pp 74-82;Structural characterization of... | |
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2015년 10월 13일 / 조회수: 601 | |
Structural characterization of calmodulin like domain of ryanodine
receptor type 1 Yonghyun
Song1†, Sunmi Kang2†, and Sunghyouk Park2* 1 Department of Biochemistry, Inha University
Hospital, College of Medicine, Inha University, Incheon, Korea 2 College of
Pharmacy, Natural Product Research Institute, Seoul National University, San
56-1 Sillim-dong, Gwanak-gu, 151-742 Seoul, Korea Received Aug 4, 2015; Revised
Sep 17, 2015; Accepted Sep 25, 2015 Abstract Ryanodine receptor (RyR) is one of the
two major Ca2+ channels in membranes of intracellular Ca2+
stores and is found in sarcoplasmic reticulum (SR), endoplasmic reticulum (ER).
RyR1 is also the major calmodulin-binding protein of sarcoplasmic reticulum
membranes. Residues 4064-4210 in the
RyR1 polypeptide chain has similar primary sequence with calmodulin (CaM) and
was designated as CaM-like domain (CaMLD). When expressed as a recombinant
peptide, CaMLD showed several CaM-like properties in previous studies. Still,
previous studies of CaMLD were focused on protein-protein interactions rather
than its own properties. Here, we studied the
expression of CaMLD and its sub-domains corresponding to each lobe of CaM in
Escherichia coli. CaMLD could be obtained only as inclusion body, and it was
refolded using urea solubilization followed by dialysis. Using spectroscopic
approaches, such as NMR, circular dichroism, and gel filtration experiment, we
found that the refolded CaMLD exists as nonspecific aggregate, even though it
has alpha helical secondary structure. In comparison, the first half of CaMLD
(R4061-4141) could be obtained as natively soluble protein with thioredoxin
fusion. After the removal of the fusion tag, it exhibited folded and helical
properties as shown by NMR and circular dichroism experiments. Its oligomeric
status was different from CaMLD, existing as dimeric form in solution. However,
the second half of the protein could not be obtained as soluble protein
regardless of fusion tag. Based on these results, we believe that CaMLD,
although similar to CaM in sequence, has quite different physicochemical
properties and that the second half of the protein renders it the aggregative
properties. Keywords RyRs, Calmodulin-like
domain, secondary structure |
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