120 of CaM could, theoretically, be spanned by the 
20-residue CaM-binding region of MLCK (RS20), an 
amino acid sequence first shown by Dr. Watterson's 
laboratory to be a high-affinity, CaM-selective bind- 
ing fragment of smooth muscle MLCK. The RS20 
CaM-binding segment appears to be in an a-helical 
conformation when bound to CaM and contains a 
potentially complementary basic-hydrophobic ami- 
no acid sequence motif Similar primary and sec- 
ondary structural motifs were detected in other 
CaM -regulated protein kinases, and synthetic pep- 
tide analogues of portions of these sequences were 
shown to bind CaM with high affinity and selectivity. 
Protein engineering and site-specific mutagene- 
sis, based on pattern analysis and perturbation of 
chemical complementarities in macromolecular in- 
teractions, were used to identify a CaM recogni- 
tion/intramolecular signal transduction region 
within a CaM-regulated protein kinase. These re- 
sults with the protein kinase were based on four in- 
terrelated sets of investigations: 1) the design and 
production in Escherichia coli of a CaM-dependent 
enzyme indistinguishable from the tissue-isolated 
enzyme in its kinetic and CaM regulatory proper- 
ties, 2) identification by deletion mutagenesis of the 
approximate boundaries of the CaM regulatory re- 
gion, 3) the design and production of chimeric en- 
zymes with the catalytic properties of an MLCK but 
the CaM recognition/regulatory properties of either 
an MLCK or a CaMPK-II (dependent on which CaM 
recognition sequence was inserted), and 4) the de- 
sign and production of a suppressor mutation in 
PUBLICATIONS 
the RS20 CaM-binding segment of the protein ki- 
nase that restores toward normal the functional ef- 
fects of point mutations in CaM. 
In summary, the results of computational, site- 
specific mutagenesis and time-resolved spectro- 
scopic studies of CaM suggest that CaM exists in a 
series of multiple, interconverting conformations in 
solution, with the addition of calcium and RS20 
peptide analogues resulting in restrictions on the 
population of conformations and their rate of inter- 
conversion. Recent structural data from other labo- 
ratories for CaM: peptide complexes, in which CaM- 
binding peptides from skeletal muscle MLCK were 
studied by nuclear magnetic resonance and neu- 
tron-scattering techniques, are consistent with this 
model. If the existence of such a multicomponent 
dynamic equilibrium is assumed, the effect of cal- 
cium and protein-protein contact may be to per- 
turb this dynamic state by restriction of the popula- 
tion of conformations, i.e., selection of preferred 
conformations by the two proteins. The results of 
the past year have enhanced the understanding of 
which features of CaM and the enzyme are impor- 
tant in coupling these intermolecular interactions 
(intermolecular signaling) to an effective perturba- 
tion of intramolecular interactions within the en- 
zyme (intramolecular signaling), with a resultant 
catalytically productive interaction between en- 
zyme and substrates. 
Dr. Watterson is also Professor of Pharmacology 
at Vanderbilt University School of Medicine. 
Books and Chapters of Books 
Hinrichsen, R.D., Watterson, D.M., and Kung, C. 1988. A mutated calmodulin from Paramecium affiects the 
Ca"*" "'^-dependent K"*" channel. In Calcium Signal and Cell Response (Yagi, K., and Miyazaki, T, Eds.). 
Tokyo: Japan Science Societies, pp 249-250. 
Articles 
Asselin, J., Phaneuf, S., Watterson, D.M., and Haiech, J. 1989. Metabolically ^^S-labeled recombinant 
calmodulin as a ligand for the detection of calmodulin-binding proteins. Anal Biochem 178:141-147. 
Chabbert, M., Kilhoffer, M.-C, Watterson, P.M. , Haiech, J., and Lami, H. 1989. Time-resolved fluorescence 
study of VU-9 calmodulin, an engineered calmodulin possessing a single tryptophan residue. Biochemis- 
try 2S:6Q95- 6098. 
Kilhoffer, M.-C, Roberts, D M., Adibi, A.O., Watterson, P.M. , and Haiech, J. 1988. Investigation of the mecha- 
nism of calcium binding to calmodulin. Use of an isofunctional mutant with a tryptophan introduced by 
site-directed mutagenesis. J Biol Chem 263:17023-17029. 
Kilhoffer, M.-C, Roberts, P.M., Adibi, A.O., Watterson, P.M. , and Haiech, J. 1989. Fluorescence characteriza- 
tion of VU-9 calmodulin, an engineered calmodulin with one tryptophan in calcium binding domain III. 
Biochemistry 28:6086-6092. 
Continued 
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