UWM Chemistry NMR facility
bar.gif

Structure Determination and Dynamics of Calmodulin in Complex with C20W and C21W, Cognate Peptides of the Plasma Membrane Calcium Pump

B. Elshorst, F. H. Försterling, M. Hennig, A. Diener, M. Maurer, P. Schulte, H. Schwalbe, C. Griesinger
J. Krebs#, H. Schmid#, T. Vorherr#, E. Carafoli#

Institute of Organic Chemistry, University of Frankfurt, D-60439 Frankfurt, Germany
# Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), CH-8092 Zürich, Switzerland
*Present Address : Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee WI 53211
bar.gif
bar.gif

Abstract

Calmodulin (CaM) is an ubiquitous Ca2+-binding protein. It plays a pivotal role in the regulation of Ca2+-dependent processes in eucaryotic cells. CaM changes its conformation upon binding of Ca2+-ions and exposes hydrophobic patches for binding target proteins, and thus transduces signalling processes. The binding regions of some of the protein targets of CaM have been identified showing amphipathic peptide sequences that upon binding to CaM formed helices like M13 (which is a synthetic peptide derived from the binding domain of the myosin light chain kinase, MLCK) and C20W, C21W or C24W corresponding to versions of different length of the CaM-binding domain of the plasma membrane Ca2+-pump2,3,4,5,6. CaM alone forms a dumbbell-like structure with a flexible linker connecting the C- and N-terminal halves. Upon binding of peptides, normally a collapsed globular structure is formed. As opposed to this we found that C20W to CaM binds only to the C-terminal domain of CaM whereas the N-terminal domain of the protein was hardly affected with the elongated structure of CaM remaining intact. The peptide C21W,on the other hand, appears to bind to both domains of calmodulin to yield a structure similar to the one of the complex with MLCK. The backbone dynamics of the CaM/C20W complex determined from 15N relaxation data allows to determine the degree of flexibility in the central helix compared with free CaM. CaM was expressed in E. coli. using minimal media, with 13C-labeled glucose and 15N-labeled ammoniumsulfate as the only carbon and nitrogen sources, respectively. C20W and C21W were synthesized and purified as described by Vorherr et al.7, and were complexed to CaM following the procedure described by Ikura et al8. Complete sequence assignments of the 13C,15N-labeled CaM/C20W and CaM/C21W peptide complexes were obtained using a number of different multinuclear NMR measurements and structural determination was achieved employing NOEs within CaM, the peptide and using interfacial NOEs. Dynamic parameters were obtained from 15N T1 and T2 relaxation time measurements and heteronuclear 1H/15N NOE measurements using the program Modelfree9. bar.gif
bar.gif
To Top of This Page NMR Home back to NMR lab on line posters To Poster-On-Line
Converted to HTML by F. Holger Försterling Feb. 2001