Defining Protein-Protein Interactions Using Site-Directed Spin-Labeling: The Binding of Protein Kinase C Substrates to Calmodulin
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- 作者：Zhihai Qin ; Stacey L. Wertz ; Jaison Jacob ; Yoko Savino ; and David S. Cafiso
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：October 15, 1996
- 年：1996
- 卷：35
- 期：41
- 页码：13272 - 13276
- 全文大小：521K
- 年卷期：v.35,no.41(October 15, 1996)
- ISSN：1520-4995
文摘
EPR spectroscopy was used to examine protein-proteininteractions between calmodulin andspin-labeled peptides based on the protein kinase C substrate domainsof the myristoylated alanine richC kinase substrate (MARCKS) and neuromodulin. When bound tocalmodulin, the C- and N-terminalends of a 25 residue MARCKS derived peptide exhibited large amplitudemotion on the nanosecond timescale and were accessible to paramagnetic agents in aqueous solution.However, residues 5-23 werehighly protected and in contact with side chains from calmodulin.These data are consistent with an
lpha.gif" BORDER=0>-helical configuration for this segment of MARCKS and withstructures that have been obtained forother calmodulin-substrate complexes. For the 17 residueneuromodulin derived peptide, which is Ca2+independent in its binding to calmodulin, oxygen collision ratesdemonstrate that one helical face of thispeptide interacts strongly with calmodulin. The data areconsistent with an interaction of this facespecifically with the C-terminal lobe of calmodulin, where this lobe iseither in an "open" or "semiopen"configuration. The EPR data also indicate that the N-terminal lobeof calmodulin is in contact with thepeptide, but that this lobe is not as strongly associated with thepeptide target. Overall, the binding pocketfor neuromodulin appears to be less compact and more dynamic than thatformed by MARCKS. Thisbehavior has not previously been seen for calmodulin substrates, and itmay play a role in the Ca2+independent binding of this class of substrates. This workdemonstrates the utility of EPR spectroscopyto define protein-protein interactions; in addition, oxygen collisionfrequencies obtained at buried sitesappear to provide information on the conformational dynamics ofproteins.
lpha.gif" BORDER=0>-helical configuration for this segment of MARCKS and withstructures that have been obtained forother calmodulin-substrate complexes. For the 17 residueneuromodulin derived peptide, which is Ca2+independent in its binding to calmodulin, oxygen collision ratesdemonstrate that one helical face of thispeptide interacts strongly with calmodulin. The data areconsistent with an interaction of this facespecifically with the C-terminal lobe of calmodulin, where this lobe iseither in an "open" or "semiopen"configuration. The EPR data also indicate that the N-terminal lobeof calmodulin is in contact with thepeptide, but that this lobe is not as strongly associated with thepeptide target. Overall, the binding pocketfor neuromodulin appears to be less compact and more dynamic than thatformed by MARCKS. Thisbehavior has not previously been seen for calmodulin substrates, and itmay play a role in the Ca2+independent binding of this class of substrates. This workdemonstrates the utility of EPR spectroscopyto define protein-protein interactions; in addition, oxygen collisionfrequencies obtained at buried sitesappear to provide information on the conformational dynamics ofproteins.