79
4. Open the result file (“rst”) and scroll down to the text “tree
with node labels.” Copy the tree into a new file and open
the file in FigTree. Identify the ancestral nodes of interest
(see Note 14) and record the node labels that identify them.
5. Search the result file for the text “node #x” (where x is the
number identifying the ancestral node) to find the maximumlikelihood ancestral sequence associated with that ancestral
node.
6. Open the alignment file in SeaView and add the ancestral protein sequences to the alignment. Edit the ancestral sequences
to remove any remaining insertions that are artifacts of the
reconstruction process (see Note 7).
Once the ancestral protein sequences have been obtained, they are
back-translated into nucleotide sequences and codon-optimized for
expression in Escherichia coli, and the genes are synthesized. The
synthetic genes are cloned into expression vectors, and the ancestral
proteins are expressed in E. coli and purified. The thermostability of
the ancestral proteins can be assessed using methods such as circular
dichroism spectroscopy, differential scanning fluorimetry (DSF), or
differential scanning calorimetry. The binding specificity of the
ancestral proteins can be assessed using methods such as isothermal
titration calorimetry (ITC), DSF, or (in some cases) fluorescence
spectroscopy. If the ancestral SBPs have high thermostability and
the desired binding specificity, they are strong candidates for circular permutation and incorporation into FRET sensor constructs.
The circular permutation of SBPs has been described by Okada
et al. [2]. It is critical to identify sites for the new N-and C- termini
that will produce a sensor with high dynamic range without negatively affecting the binding affinity or stability of the protein.
1. Using a crystal structure or homology model of the ancestral
SBP (created using the Phyre2 server [27], for example), select
the positions of the new N- and C-termini. The new termini
must be located on different lobes of the SBP to maximize the
change in their relative positions due to the ligand-induced
conformational change. This can be achieved by deleting a section of the protein that links the two lobes (i.e., a hinge strand)
(see Note 15). The result is a theoretical protein with the original N- and C-termini, and additional N*- and C*-termini, i.e.,
two protein fragments.
2. Design a linker sequence to fuse the original N- and C-termini
of the SBP. Each repeat of a flexible (GGS)n linker is approximately 11.4 Å in length. Measure the distance between the
N- and C-termini of the SBP; the linker should have enough
(GGS)n repeats to bridge this distance.
3.6 Characterization
of Ancestral Proteins
3.7 Design
of Circularly
Permuted SBPs
Improving FRET Sensors by Ancestral Gene Resurrection
4. Open the result file (“rst”) and scroll down to the text “tree
with node labels.” Copy the tree into a new file and open
the file in FigTree. Identify the ancestral nodes of interest
(see Note 14) and record the node labels that identify them.
5. Search the result file for the text “node #x” (where x is the
number identifying the ancestral node) to find the maximumlikelihood ancestral sequence associated with that ancestral
node.
6. Open the alignment file in SeaView and add the ancestral protein sequences to the alignment. Edit the ancestral sequences
to remove any remaining insertions that are artifacts of the
reconstruction process (see Note 7).
Once the ancestral protein sequences have been obtained, they are
back-translated into nucleotide sequences and codon-optimized for
expression in Escherichia coli, and the genes are synthesized. The
synthetic genes are cloned into expression vectors, and the ancestral
proteins are expressed in E. coli and purified. The thermostability of
the ancestral proteins can be assessed using methods such as circular
dichroism spectroscopy, differential scanning fluorimetry (DSF), or
differential scanning calorimetry. The binding specificity of the
ancestral proteins can be assessed using methods such as isothermal
titration calorimetry (ITC), DSF, or (in some cases) fluorescence
spectroscopy. If the ancestral SBPs have high thermostability and
the desired binding specificity, they are strong candidates for circular permutation and incorporation into FRET sensor constructs.
The circular permutation of SBPs has been described by Okada
et al. [2]. It is critical to identify sites for the new N-and C- termini
that will produce a sensor with high dynamic range without negatively affecting the binding affinity or stability of the protein.
1. Using a crystal structure or homology model of the ancestral
SBP (created using the Phyre2 server [27], for example), select
the positions of the new N- and C-termini. The new termini
must be located on different lobes of the SBP to maximize the
change in their relative positions due to the ligand-induced
conformational change. This can be achieved by deleting a section of the protein that links the two lobes (i.e., a hinge strand)
(see Note 15). The result is a theoretical protein with the original N- and C-termini, and additional N*- and C*-termini, i.e.,
two protein fragments.
2. Design a linker sequence to fuse the original N- and C-termini
of the SBP. Each repeat of a flexible (GGS)n linker is approximately 11.4 Å in length. Measure the distance between the
N- and C-termini of the SBP; the linker should have enough
(GGS)n repeats to bridge this distance.
3.6 Characterization
of Ancestral Proteins
3.7 Design
of Circularly
Permuted SBPs
Improving FRET Sensors by Ancestral Gene Resurrection