In addition, the interaction between CaM and calcium is impacted by cooperativity. The calcium concentration at which binding to CaM occurs (and fluorescence decreases) is referred to as the K d and determined by the affinity of CaM to calcium. The transition from bright to dim fluorescence occurs over a particular concentration range of calcium. However, when enough calcium is added to a solution of inverse pericam, CaM and M13 interact, disrupting the conformation and, as a result, the fluorescence of EYFP. In the absence of calcium, this EYFP exhibits strong fluorescence.
Within inverse pericam, M13 and CaM are located at opposite ends, surrounding a permuted (i.e., rearranged) version of EYFP. (B) In the absence of Ca 2+, IPC fluoresces yellow and in the presence of Ca 2+ fluorescence is quenched. (A) The EYFP gene within IPC is mutated such that the C and N termini are re-organized then flanked by M13 and CaM. This conditional specificity for M13 binding is enabled by the change in confirmation of CaM when bound to calcium. Calmodulin has many ligands that it binds only in the presence of calcium ion, including the peptide fragment M13. The other key component of inverse pericam is the protein calmodulin (CaM), a natural calcium sensor that is present in all eukaryotes. It has since been exploited as a genetically encodable reporter and mutagenized to vary its excitation and emission spectra. GFP is naturally produced by jellyfish and was cloned into other organisms in the early 1990’s. The fluorescent component of IPC is an enhanced yellow fluorescent protein (abbreviated EYFP), one of the many derivatives of green fluorescent protein (GFP). Today you will familiarize yourself with the recombinant protein IPC and its constituent parts. 2.3 Part 3: Examine IPC structural elements.2.2 Part 2: Identify IPC sequence features.2.1 Part 1: Review IPC reference article.
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