Abstract
Protein splicing is the post translational modification by which an internal peptide, or intein, separates itself from the surrounding residues, or exteins. In the third step of the mechanism, asparagine cyclization, coupled to peptide bond cleavage, cleaves the intein from the exteins. We have studied this cyclization both experimentally and computationally. In the lab, we use a model peptide assay in which the cyclization of Asn is coupled to fluorescence. Cleavage via Asn cyclization is much faster than via Gln cyclization. High temperature, low dielectric constant, and high pH promote cyclization. We also have observed cleavage after Asp, but only at low pH. Computationally, we used molecular dynamics simulations, which suggest that the Asn side chain more frequently samples optimal attack conformations than a Gln side chain. Density functional theory and nudged elastic band simulations confirm the side chain nitrogen, not the oxygen, is the nucleophile and that nitrogen deprotonation is likely necessary for the attack to occur. The activation barriers of both the deprotonation and cyclization are being calculated to determine the rate limiting step for the mechanism. Simulations of peptides containing Asp suggest that cyclization occurs via a different mechanism than that of the native Asn and Gln residues. This material is based upon work supported by the National Science Foundation under grant MCB-1244089 and by the Camille and Henry Dreyfus Foundation.
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