TRIPLE-RESONANCE MULTIDIMENSIONAL NMR-STUDY OF CALMODULIN COMPLEXED WITH THE BINDING DOMAIN OF SKELETAL-MUSCLE MYOSIN LIGHT-CHAIN KINASE - INDICATION OF A CONFORMATIONAL CHANGE IN THE CENTRAL HELIX
TRIPLE-RESONANCE MULTIDIMENSIONAL NMR-STUDY OF CALMODULIN COMPLEXED WITH THE BINDING DOMAIN OF SKELETAL-MUSCLE MYOSIN LIGHT-CHAIN KINASE - INDICATION OF A CONFORMATIONAL CHANGE IN THE CENTRAL HELIX
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DOI：
10.1021/bi00236a024
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发表时间：
1991-06-04
期刊：
影响因子：
2.9
通讯作者：
BAX, A
中科院分区：
文献类型：
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作者：
IKURA, M;KAY, LE;BAX, A
利用异核三维和四维核磁共振实验获得了钙调蛋白与兔骨骼肌肌球蛋白轻链激酶钙调蛋白结合域（残基577-602）对应的26个残基合成肽（M13）配合的钙调蛋白中H-1、C-13和N-15骨干化学位移配位。与无肽钙调素的化学位移值的比较[Ikura， M., Kay， L. E., & Bax， A. (1990) Biochemistry 29, 4659-4667]表明，M13肽的结合诱导了大量的化学位移变化，而这些变化并不局限于蛋白质的一个特定区域。最大的变化发生在Ca2+结合位点I的第一个螺旋（E11-E14），中央螺旋的n端部分（M72-D78）和Ca2+结合位点IV的第二个螺旋（F141-M145）。主链NOE连通性分析表明，残基75-77在与M13络合后由α -螺旋构象转变为扩展构象。这些残基相对于无m13的钙调素的c - α和羰基化学位移的上场变化以及氢交换实验表明，残基75-82的酰胺质子与溶剂进行了快速交换（k(exch) bbb10 s-1， pH值为7,35℃），支持了这种构象变化。位点I的第一螺旋和位点IV的c端螺旋的二级结构没有变化。与M13络合后，位点III第二螺旋末端的残基T110、L112、G113和E114的酰胺交换率显著降低。
Heteronuclear 3D and 4D NMR experiments have been used to obtain H-1, C-13, and N-15 backbone chemical shift assignments in Ca2+-loaded calmodulin complexed with a 26-residue synthetic peptide (M13) corresponding to the calmodulin-binding domain (residues 577-602) of rabbit skeletal muscle myosin light-chain kinase. Comparison of the chemical shift values with those observed in peptide-free calmodulin [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667] shows that binding of M13 peptide induces substantial chemical shift changes that are not localized in one particular region of the protein. The largest changes are found in the first helix of the Ca2+-binding site I (E11-E14), the N-terminal portion of the central helix (M72-D78), and the second helix of the Ca2+-binding site IV (F141-M145). Analysis of backbone NOE connectivities indicates a change from alpha-helical to an extended conformation for residues 75-77 upon complexation with M13. This conformational change is supported by upfield changes in the C-alpha and carbonyl chemical shifts of these residues relative to M13-free calmodulin and by hydrogen-exchange experiments that indicate that the amide protons of residues 75-82 are in fast exchange (k(exch) > 10 s-1 at pH 7, 35-degrees-C) with the solvent. No changes in secondary structure are observed for the first helix of site I or the C-terminal helix of site IV. Upon complexation with M13, a significant decrease in the amide exchange rate is observed for residues T110, L112, G113, and E114 at the end of the second helix of site III.