+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Defining protein-protein interactions using site-directed spin-labeling: the binding of protein kinase C substrates to calmodulin



Defining protein-protein interactions using site-directed spin-labeling: the binding of protein kinase C substrates to calmodulin



Biochemistry 35(41): 13272



EPR spectroscopy was used to examine protein-protein interactions between calmodulin and spin-labeled peptides based on the protein kinase C substrate domains of the myristoylated alanine rich C kinase substrate (MARCKS) and neuromodulin. When bound to calmodulin, the C- and N-terminal ends of a 25 residue MARCKS derived peptide exhibited large amplitude motion on the nanosecond time scale and were accessible to paramagnetic agents in aqueous solution. However, residues 5-23 were highly protected and in contact with side chains from calmodulin. These data are consistent with an alpha-helical configuration for this segment of MARCKS and with structures that have been obtained for other calmodulin-substrate complexes. For the 17 residue neuromodulin derived peptide, which is Ca2+ independent in its binding to calmodulin, oxygen collision rates demonstrate that one helical face of this peptide interacts strongly with calmodulin. The data are consistent with an interaction of this face specifically with the C-terminal lobe of calmodulin, where this lobe is either in an "open" or "semiopen" configuration. The EPR data also indicate that the N-terminal lobe of calmodulin is in contact with the peptide, but that this lobe is not as strongly associated with the peptide target. Overall, the binding pocket for neuromodulin appears to be less compact and more dynamic than that formed by MARCKS. This behavior has not previously been seen for calmodulin substrates, and it may play a role in the Ca2+ independent binding of this class of substrates. This work demonstrates the utility of EPR spectroscopy to define protein-protein interactions; in addition, oxygen collision frequencies obtained at buried sites appear to provide information on the conformational dynamics of proteins.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 008424212

Download citation: RISBibTeXText

PMID: 8873591

DOI: 10.1021/bi961747y


Related references

Mapping peptide-protein interactions using site directed spin-labeling The binding of protein kinase C substrates to calmodulin. Biophysical Journal 70(2 Part 2): A59, 1996

Membrane structure of protein kinase C and calmodulin binding domain of myristoylated alanine rich C kinase substrate determined by site-directed spin labeling. Biochemistry 35(9): 2917-2925, 1996

Estimation of binding parameters for the protein-protein interaction using a site-directed spin labeling and EPR spectroscopy. European Biophysics Journal 37(4): 483-493, 2008

Inter- and intra-molecular distances determined by EPR spectroscopy and site-directed spin labeling reveal protein-protein and protein-oligonucleotide interaction. Biological Chemistry 385(10): 913-920, 2004

Site directed mutagenesis of a calmodulin dependent protein kinase effects of chimeric calmodulin binding segments on calmodulin recognition and activity. Biophysical Journal 57(2 Part 2): 425A, 1990

Active site-directed inhibition of Ca2+/calmodulin-dependent protein kinase type II by a bifunctional calmodulin-binding peptide. Proceedings of the National Academy of Sciences of the United States of America 85(14): 4991-4995, 1988

Identification of the calmodulin-binding domain of neuron-specific protein kinase C substrate protein CAP-22/NAP-22. Direct involvement of protein myristoylation in calmodulin-target protein interaction. Journal of Biological Chemistry 274(17): 11848-11853, 1999

Interactions of the GM2 activator protein with phosphatidylcholine bilayers: a site-directed spin-labeling power saturation study. Biophysical Journal 97(5): 1436-1444, 2009

C2 Domain of Protein Kinase Ca: Elucidation of the Membrane Docking Surface by Site-Directed Fluorescence and Spin Labeling. Biochemistry (American Chemical Society) 42(5): 54-65, 2003

C2 domain of protein kinase C alpha: elucidation of the membrane docking surface by site-directed fluorescence and spin labeling. Biochemistry 42(5): 1254-1265, 2003

A surface plasmon resonance study of the interactions between the component subunits of protein kinase CK2 and two protein substrates, casein and calmodulin. Molecular and Cellular Biochemistry 227(1-2): 31-36, 2001

A surface plasmon resonance study of the interactions between the component subunits of protein kinase CK2 and two protein substrates, casein and calmodulin. Molecular and Cellular Biochemistry 227(1-2): 31-36, 2001

Affinity labeling of the ATP-binding site of type II calmodulin-dependent protein kinase by 5'-p-fluorosulfonylbenzoyl adenosine. Archives of Biochemistry and Biophysics 267(2): 467-473, 1988

CW EPR and DEER Methods to Determine BCL-2 Family Protein Structure and Interactions: Application of Site-Directed Spin Labeling to BAK Apoptotic Pores. Methods in Molecular Biology 1877: 257-303, 2019

Membrane-bound structure of the N-terminal binding domain of the pp60src protein determined by site-directed spin labeling. Biophysical Journal 74(2 Part 2): A302, 1998