Expressed Protein Ligation

We use EPL approaches to insert precise histone modifications into the structured nucleosome core. We have shown that core modifications play roles in the regulating the mobility of nucleosomes on DNA sequences, and in facilitating chromatin remodeling by DNA repair systems.

Multistep Chemical Ligation

Modifications near the middle of the histone sequence, like acetylation of H3-Lys56, are accessible through total synthesis of histone proteins in solution. We have successfully synthesized histone H3 using a multi-step chemical ligation and desulfurization strategy. This strategy allows introduction of multiple modifications throughout the histone sequence.

Hybrid Phase Chemical Ligation

Sequential chemical ligation works, but is not very efficient and requires building challenging, long peptides to minimize the number of steps.  We developed a solid phase approach for total histone synthesis, but found that yields were very low.  We are now working on a hybrid phase approach for histone synthesis that mixes initial solid phase steps with solution phase ligation for increased yields with minimal effort and maximum efficiency.

Fmoc-SPPS of Peptide Thioesters

In order to use chemical ligation to generate histones, we need peptide thioesters that cover the histone sequence. This has proven challenging using existing techniques.  We have been exploring improvements to the elegant Dawson N-acylurea approach that are compatible with the long and glycine-rich peptides required for our work. We find that orthogonal protection of the linker to generate an Fmoc-Dbz(Alloc) base resin is efficient and compatible with most amino acids used at ligation junctions.