Biophysical characterization of the interaction of p21 with calmodulin: A mechanistic study Qiaoyun Shi, Xiaohui Wang, Jinsong Ren ⁎ Division of Biological Inorganic Chemistry, State Key laboratory of Rare Earth Resources Utilization, Graduate School of the Chinese Academy of Sciences, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China abstract article info Article history: Received 25 August 2008 Received in revised form 15 September 2008 Accepted 15 September 2008 Available online 20 September 2008 Keywords: p21 Calmodulin Dansyl Tryptophan Fluorescence quenching Circular dichroism p21 is a protein with important roles in cell proliferation, cell cycle regulation and apoptosis. Several studies have demonstrated that its intracellular localization plays an important role in the functional regulation and binding of calmodulin favors its nuclear translocation. However, the detail mechanism of the interaction with p21 and calmodulin is not well understood. In this report, peptides derived from the C-terminal of p21 that cover the binding domain of calmodulin were used to investigate the association of p21 with calmodulin. We found p21 141–164 interaction with Ca 2+ -saturated dansyl-labelled calmodulin caused a significant increase in dansyl fluorescence intensity and a blue shift of the maximum emission from 510 to 475 nm. The Trp fluorescence intensities of mutated p21 141–164 peptides (F150W, Y151Wand F159W) increased upon binding to Ca 2+ -saturated calmodulin and fluorescence maxima were blue shifted from 350 nm to 330 nm. The results suggested p21 141–164 is most likely buried in the hydrophobic binding tunnel of calmodulin. Both dansyl and Trp fluorescence titrations generated dissociation constants around 0.1 μM and a stoichiometry of 1:1, which was further confirmed by nondenaturing gel band shift electrophoresis. Fluorescence titrations and Trp fluorescence quenching results indicated electrostatic interaction is involved in this association. Upon binding to calmodulin, p21 141–164 remained largely unstructured and showed only about 15% α-helix. In contrast to other calmodulin binding peptide, the dominant force in the association of p21 141–164 with calmodulin may be electrostatic interaction. Our results would be helpful for understanding the molecular details of p21 and calmodulin interaction. © 2008 Elsevier B.V. All rights reserved. 1. Introduction p21 is a protein with important roles in cell proliferation, cell cycle regulation, differentiation, senescence, and apoptosis [1–4]. It exhibits different functions in nuclear and cytoplasmic compartments [5]. Nuclear p21 induces a cycle arrest and inhibits DNA synthesis, functioning as a tumor suppressor [6,7], while cytoplasmic p21 has proliferation, anti-apoptosis and motility functions [8,9]. Calmodulin (CaM) is a Ca 2+ binding protein and acts as transducer of the intracellular Ca 2+ signal [10,11]. When bound to Ca 2+ , CaM is able to bind to CaM-binding proteins and regulate their activity [12–14]. Agell et al. has demonstrated that Ca 2+ saturated CaM can bind to the carboxyl-terminal domain of p21 [15] and that CaM binding favors the nuclear accumulation of p21 [16]. However, detail mechanism of this interaction is not clear, partly due to the lack of structural information. Herein, peptides derived from the C-terminal domain of p21 that cover the binding domain of the CaM were used to investigate the association of p21 with CaM. We found that p21 141–164 formed a 1:1 complex with Ca 2+ -saturated CaM and the dissociation was around 0.1 μM. Surprisingly, p21 141–164 remained largely unstructured and showed only about 15% α-helix when bound to CaM. The results suggest the dominant force in the association of p21 141–164 with CaM may be electrostatic interaction. To the best of our knowledge, this is the first report that systematically characterizes the association of p21 with CaM. 2. Materials and methods 2.1. Peptide synthesis and protein preparation Four peptides encompassing the putative CaM-binding domain of the C-terminal of p21 were chemically synthesized by the CL Bio- scientific CO. LTD. The sequences were as follow: p21 141–164 , KRRQTSMTDFYHSKRRLIFSKRKP; F150W, KRRQTSMTDWYHSKRR- LIFSKRKP, substituting F150 of p21 141–164 with W; Y151W, KRRQTSMTDFWHSKRRLIFSKRKP, substituting Y151 of p21 141–164 with W; F159W, KRRQTSMTDFYHSKRRLIWSKRKP, substituting F159 of p21 141–164 with W. The purity of peptides was 95% according to high pressure liquid chromatography. The concentrations of all the Biophysical Chemistry 138 (2008) 138–143 ⁎ Corresponding author. Tel.: +86 431 8526 2625; fax: +86 431 85262656. E-mail address: jren@ciac.jl.cn (J. Ren). 0301-4622/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bpc.2008.09.012 Contents lists available at ScienceDirect Biophysical Chemistry journal homepage: http://www.elsevier.com/locate/biophyschem