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Research Paper

An Improved Surface Plasmon Resonance Method for Determining the Molecular Basis of the Calcium-Dependent Interactions of ALG-2

Lalita Subramanian, Theresa M. Walker, Jennifer C. Takach and Arthur S. Polans

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Elucidation of the molecular dynamics involved in calcium-dependent proteinprotein interactions has often proved technically challenging. We have successfully used surface plasmon resonance (SPR) technology in combination with a novel method involving the immobilization of streptavidin binding peptide (SBP)-tagged proteins to streptavidin-coated chips to investigate the interactions of Apoptosis Linked Gene-2 (ALG-2) with the N-terminal region of annexin XI. ALG-2 is a calcium binding protein of the penta-EF hand family, with identified high affinity Ca2+-binding sites, putative binding partners as well as suggested regions of target recognition based on its crystal structure. The new tandem affinity tag vector allowed the purification of the expressed annexin XI using an N-terminal calmodulin binding peptide (CBP)-tag while the SBP tag allowed the directional tethering to the streptavidin-coated gold chip to create a homogeneous surface for SPR measurements. The interaction of ALG-2 with annexin XI was found to be Ca2+-dependent requiring both high affinity EFhands, EF1 and EF3, to be functional. Further, the hydrophobic pocket observed in the crystal structure but not the N-terminal region, was identified as the common recognition site for two putative target molecules, annexin XI and Alix/AIP1. This method was found suitable for application in two different SPR instruments and particularly lends itself to the analysis of Ca2+-dependent interactions.

Authors

Lalita Subramanian

Theresa M. Walker

Jennifer C. Takach

This is an open-access article

 Download PDF

If the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.