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J. Biol. Chem., Vol. 278, Issue 44, 43764-43769, October 31, 2003

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Conformational Changes in the Ca²⁺-regulatory Region from Soybean Calcium-dependent Protein Kinase-

FLUORESCENCE RESONANCE ENERGY TRANSFER STUDIES^*

Aalim M. Weljie, Kindal M. Robertson, and Hans J. Vogel

From the Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Alberta T2N 1N4, Canada

Calcium-dependent protein kinases are key proteins involved in plant and protozoal Ca²⁺ signaling. These unique molecules include a calcium regulatory calmodulin-like domain (CLD), which binds to another small regulatory domain named the junction domain (JD). Both CLD and JD are part of the same polypeptide as the protein kinase domain. The CLD consists of N- and C-terminal lobes, each having two helix-loop-helix Ca²⁺-binding motifs. In this study, fluorescence resonance energy transfer using a series of Trp and Cys site-directed mutants was undertaken to probe the relative motions of the two lobes of CLD between the apo- and Ca²⁺-saturated forms, as well as bound to a peptide encoding the JD sequence. Using an IAEDANS-modified Cys, a total of 15 Trp Cys distances were measured in these three states from the five donor-acceptor combinations F334W-Cys⁴³⁶, L371W-Cys⁴³⁶, L403W-Cys⁴³⁶, F334W-L403C, and L371W-L403C. Consistent with recently reported NMR diffusion measurements and with ¹H,¹⁵N heteronuclear correlation NMR spectra, the distances derived from fluorescence resonance energy transfer measurements in apoCLD indicate partial unfolding and a subsequent contraction on binding Ca²⁺, which is maintained on addition of the JD peptide. Interpretation of the distances suggests that the Ca²⁺-saturated form is open with the two lobes sitting side-by-side although highly flexible. The transition to the JD-CLD state appears to be accompanied by a rotation of the N- and C-terminal domains with respect to each other inducing a slightly more closed overall complex. The observed differences between the behavior of CLD and the well studied related protein calmodulin are likely because of different physiological requirements for activation in vivo.

Received for publication, June 25, 2003 , and in revised form, August 26, 2003.

^* This work supported by an operating grant from the National Sciences and Engineering Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of an Alberta Heritage Foundation for Medical Research Studentship award.

Holds a scientist award from Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed. Tel.: 403-220-6006; Fax: 403-289-9311; E-mail: vogel{at}ucalgary.ca.

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				A. M. Weljie and H. J. Vogel Unexpected Structure of the Ca2+-regulatory Region from Soybean Calcium-dependent Protein Kinase-{alpha} J. Biol. Chem., August 20, 2004; 279(34): 35494 - 35502. [Abstract] [Full Text] [PDF]