Control of and imaging techniques for protein nanoscaffolds
Described herein are constructs, compositions and methods for precise in vivo imaging of the structures and dynamics of protein-based scaffolds with and without their designated cargos.
1. A self-assembling protein comprising (a) a first fusion protein comprising a pfam00936 domain of Scaffolds Formed by BMC-Shell proteins (ScaF) linked to a first synthetic zipper domain via a flexible peptide linker, wherein the pfam00936 domain has an amino acid sequence with at least 90% sequence identity to any of SEQ ID NO:1-4; and (b) a second fusion protein comprising a second synthetic zipper domain linked to a flexible linker, wherein:
the first synthetic zipper domain and the second synthetic zipper domain are coiled-coil protein domains that noncovalently bind together to form a heterodimer; and
the flexible linker is configured to link to a cargo protein and the flexible linker comprises at least 60% glycine residues, serine residues, or a combination of glycine and serine residues.
2. The self-assembling protein of claim 1 , wherein the flexible peptide linker is a 3 to 20 amino acid peptide.
3. The self-assembling protein of claim 1 , wherein the first synthetic zipper has an amino acid sequence with at least 90% sequence identity to one of SEQ ID NO:5 or 6, and wherein the second synthetic zipper has an amino acid sequence with at least 90% sequence identity to the other of SEQ ID NO:5 or 6 and binds to the first synthetic zipper.
4. The self-assembling protein of claim 1 , wherein the pfam00936 domain (ScaF), the cargo protein, or a combination thereof each have a covalently linked marker or reporter.
5. A self-assembling protein comprising:
(a) a pfam00936 domain of Scaffolds Formed by BMC-Shell proteins (ScaF) linked to a first synthetic zipper domain via a flexible peptide linker, wherein the pfam00936 domain has an amino acid sequence with at least 90% sequence identity to any of SEQ ID NO:1-4 and the first synthetic zipper domain has an amino acid sequence with at least 90% sequence identity to either of SEQ ID NO:5 or 6; and (b) a cargo protein linked to a second synthetic zipper domain via a flexible linker, wherein the second synthetic zipper has an amino acid sequence with at least 90% sequence identity to the other of SEQ ID NO:5 or 6, wherein the flexible linker comprises at least 60% glycine residues, serine residues, or a combination of glycine and serine residues.
6. The self-assembling protein of claim 5 , wherein the flexible peptide linker is a 3 to 20 amino acid peptide.
7. The self-assembling protein of claim 1 , wherein the at least one of the cargo proteins is a fluorescent protein.