1. Calcium-Induced Conformational Switching of Paramecium Calmodulin Provides Evidence for Domain Coupling Jaren et al. Biochemistry 2002, 41,
Susan Ahrens
February 24, 2004

2. Outline
Introduction
Methods
Data
Conclusions

3. Calmodulin Function Ca2+ binding protein
Used in many pathways in signal transduction
Functions in gene regulation, protein synthesis, ion channel function, cell motility and chemotaxis.
Target proteins include
kinases
cyclases

4. Calmodulin Structure

5. CaM Structure
Ca2+ binding sites I and
II of PCaM

6. Methods NMR sample preparation NMR Instrumentation
Grown in enriched media and purified through chromatographic techniques
Apo vs. Ca2+ saturated samples
NMR Instrumentation
Spectra recorded on Varian INOVA spectrometers a t 500, 600, and 750 MHz (1H)
DSS in D2O used for standard

7. NMR Assignments for 13C, 15N-labeled PCaM
HN, N, Ca, Cb, and C’-pulse sequence
1) 1H-15N HSQC
2) 1H-13C Constant time HSQC
3) CBCA(CO)NH
4) HNCACB
5) HNCO
6) HN(CA)CO
7) CTSL-HCANH
8) 15N-edited 3D NOESY

8. Ca2+ and Cd2+ titrations Monitored by NMR Ca2+ Cd2+
15N-1H HSQC NMR using INOVA 500
1024 points, 256 increments, and 8 transients per increment
Spectral width 14 ppm for 1H and 49 ppm for 15N
Cd2+
9.4 T Bruker instrument at 88.7 MHz
Spectrum generated from 23,400 scans
Spectral width 450 ppm

9. PCaM HSQC Spectra Apo PCaM PCaM at Ca2+/CaM ratio of 1.93
Ca2+-saturated PCaM
Ca2+-saturated in RCaM

10. Cation titration of sites III and IV of
PCaM monitored by changes in peak
intensities

11. Line broadening behavior of the N-domain resonances as shown
By 1H cross sections of 1H-15N HSQC spectra

12. Ca2+ titration of sites I
and II monitored by
changes in frequency
and 1H-15N HSQC
resonance intensity

13. Ca2+-dependent behavior
of the E78 resonance as
monitored by 1H-15N HSQC
NMR

14. Ca2+-dependent behavior of interdomain linker residues
monitored by 1H-15N HSQC NMR

15. Calmodulin Structure

16. Conclusions
Linker residues exhibit a domain-specific, Ca2+-dependent response to binding in all four sites
The interdomain linker serves as a conformational switch that couples the Ca2+-dependant properties of the two domains in CaM and is activated upon ligation of sites III and IV in the C-domain
Residue specific responses in CaM?