Abstract
Structural studies of the calmodulin-dependent protein kinase I have shown how the calmodulin-binding domain and autoinhibitory domain interact with the active sites of the enzyme. In this work, we have studied the interaction in solution of two synthetic short and long (22- and 37-residue) peptides representing the binding and autoinhibitory domains of CaMKI with Ca 2+-CaM using CD, NMR, and EPR spectroscopy. Both peptides adopt α-helical structure when bound to Ca2+-CaM, as detected by CD spectroscopy. Cadmium-113 NMR showed that both peptides induced cooperativity in metal ion binding between the two lobes of the protein. To directly observe the effect of the peptides upon CaM in solution, biosynthetically isotope labeled [methyl-13C-Met]CaM was prepared and studied by 1H, 13C NMR. The relaxation effects of two nitroxide spin-labeled derivatives of the short peptide showed the N-terminal portion of the CaM-binding domain interacting with the C-lobe of CaM, while the C-lobe of the peptide binds to the N-lobe of CaM. Our results are consistent with Trp303 and Met316 acting as the anchoring residues for the C- and N-lobes of CaM, respectively. The NMR spectra of the long peptide showed further differences, suggesting that additional interactions may exist between the autoinhibitory domain and CaM.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 192-206 |
| Number of pages | 15 |
| Journal | Archives of Biochemistry and Biophysics |
| Volume | 421 |
| Issue number | 2 |
| DOIs | |
| State | Published - Jan 15 2004 |
| Externally published | Yes |
Fingerprint
Keywords
- Calmodulin
- Calmodulin-dependent protein kinase I
- Circular dichroism
- Electron paramagnetic resonance
- Nuclear magnetic resonance
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Spectroscopic characterization of the calmodulin-binding and autoinhibitory domains of calcium/calmodulin-dependent protein kinase I. / Yuan, Tao; Gomes, Aldrin V; Barnes, Junor A.; Hunter, Howard N.; Vogel, Hans J.
In: Archives of Biochemistry and Biophysics, Vol. 421, No. 2, 15.01.2004, p. 192-206.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Spectroscopic characterization of the calmodulin-binding and autoinhibitory domains of calcium/calmodulin-dependent protein kinase I
AU - Yuan, Tao
AU - Gomes, Aldrin V
AU - Barnes, Junor A.
AU - Hunter, Howard N.
AU - Vogel, Hans J.
PY - 2004/1/15
Y1 - 2004/1/15
N2 - Structural studies of the calmodulin-dependent protein kinase I have shown how the calmodulin-binding domain and autoinhibitory domain interact with the active sites of the enzyme. In this work, we have studied the interaction in solution of two synthetic short and long (22- and 37-residue) peptides representing the binding and autoinhibitory domains of CaMKI with Ca 2+-CaM using CD, NMR, and EPR spectroscopy. Both peptides adopt α-helical structure when bound to Ca2+-CaM, as detected by CD spectroscopy. Cadmium-113 NMR showed that both peptides induced cooperativity in metal ion binding between the two lobes of the protein. To directly observe the effect of the peptides upon CaM in solution, biosynthetically isotope labeled [methyl-13C-Met]CaM was prepared and studied by 1H, 13C NMR. The relaxation effects of two nitroxide spin-labeled derivatives of the short peptide showed the N-terminal portion of the CaM-binding domain interacting with the C-lobe of CaM, while the C-lobe of the peptide binds to the N-lobe of CaM. Our results are consistent with Trp303 and Met316 acting as the anchoring residues for the C- and N-lobes of CaM, respectively. The NMR spectra of the long peptide showed further differences, suggesting that additional interactions may exist between the autoinhibitory domain and CaM.
AB - Structural studies of the calmodulin-dependent protein kinase I have shown how the calmodulin-binding domain and autoinhibitory domain interact with the active sites of the enzyme. In this work, we have studied the interaction in solution of two synthetic short and long (22- and 37-residue) peptides representing the binding and autoinhibitory domains of CaMKI with Ca 2+-CaM using CD, NMR, and EPR spectroscopy. Both peptides adopt α-helical structure when bound to Ca2+-CaM, as detected by CD spectroscopy. Cadmium-113 NMR showed that both peptides induced cooperativity in metal ion binding between the two lobes of the protein. To directly observe the effect of the peptides upon CaM in solution, biosynthetically isotope labeled [methyl-13C-Met]CaM was prepared and studied by 1H, 13C NMR. The relaxation effects of two nitroxide spin-labeled derivatives of the short peptide showed the N-terminal portion of the CaM-binding domain interacting with the C-lobe of CaM, while the C-lobe of the peptide binds to the N-lobe of CaM. Our results are consistent with Trp303 and Met316 acting as the anchoring residues for the C- and N-lobes of CaM, respectively. The NMR spectra of the long peptide showed further differences, suggesting that additional interactions may exist between the autoinhibitory domain and CaM.
KW - Calmodulin
KW - Calmodulin-dependent protein kinase I
KW - Circular dichroism
KW - Electron paramagnetic resonance
KW - Nuclear magnetic resonance
UR - http://www.scopus.com/inward/record.url?scp=0347931868&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0347931868&partnerID=8YFLogxK
U2 - 10.1016/j.abb.2003.11.012
DO - 10.1016/j.abb.2003.11.012
M3 - Article
C2 - 14984199
AN - SCOPUS:0347931868
VL - 421
SP - 192
EP - 206
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
SN - 0003-9861
IS - 2
ER -