31
present less of a risk for aggregation or degradation, although the
opposite may be true. In any case, a binding mutation near one of
the termini gives one the freedom to experiment with both short
and long duplicate segments.
For a binding mutation near the C-terminus, the duplicated segment ends at the C-terminus (Fig. 1a). It begins at a position
N-terminal to the binding mutation and this position is chosen to
be a surface loop or turn. The reason is that this loop becomes the
permutation site of the N′-fold, i.e., the location at which the polypeptide chain is broken and new N- and C-termini are generated.
Interrupting an alpha helix, beta strand, or buried hydrophobic
region is expected to be more destabilizing than disrupting a surface loop, although there are examples of successful permutation
sites at the former locations [5–10]. The same considerations apply
to the case where the binding mutation is near the N-terminus,
except the duplicated segment begins with the N-terminus and ends
at a surface loop C-terminal to the binding mutation (Fig. 1a).
Inability to find a stable circular permutant (CP) is the most
common failure point in the AFF protocol. Permutation almost
always destabilizes a protein, and there is no reliable method for
predicting the extent of destabilization for a given permutation site.
The CP needs to be at least marginally stable (ΔG unfold ≥ 2–3 kcal/mol),
with more stable CPs requiring less optimization (see Subheading 5).
Our approach for selecting permutation sites is to choose the first
three to four surface loops either N-terminal or C-terminal to the
binding mutation, depending on whether the binding mutation is
closer to the C-terminus or N-terminus, respectively. Loops that are
close to the binding/active site should be avoided for functional
reasons, although xylanase [9] and beta lactamase [11] were permuted at several loops proximal to their active sites without major
loss of activity. Fortunately, all but the smallest POIs will have many
loops from which to choose and at least one will usually be stable
and functional enough for the AFF design. For example, RBP (277
amino acids) has 13 surface loops (Fig. 2a). We created CPs at eight
of these loops and all were stable, soluble, and functional. All were
destabilized compared to wild-type (WT) RBP, however, and this
finding demonstrates the advantage of starting with the most stable
variant of the POI available.
The linker functions to physically bridge the original N- and
C-termini of the POI. It effectively becomes a new surface loop of
the CP. As such, the amino acid sequence should be hydrophilic
and flexible enough to not impose any new constraints on the
protein structure. We base our linkers on Gly/Ala/Ser repeats,
although more advanced design criteria have been discussed
[9, 11–15]. With regard to linker length, a rule of thumb is to
measure the N-to-C distance (C α –C α ) from the structure “as the
3.2 Identify
a Circular Permutation
Site
3.3 Design
the Peptide Linker
Engineering Allosteric Protein Switches
present less of a risk for aggregation or degradation, although the
opposite may be true. In any case, a binding mutation near one of
the termini gives one the freedom to experiment with both short
and long duplicate segments.
For a binding mutation near the C-terminus, the duplicated segment ends at the C-terminus (Fig. 1a). It begins at a position
N-terminal to the binding mutation and this position is chosen to
be a surface loop or turn. The reason is that this loop becomes the
permutation site of the N′-fold, i.e., the location at which the polypeptide chain is broken and new N- and C-termini are generated.
Interrupting an alpha helix, beta strand, or buried hydrophobic
region is expected to be more destabilizing than disrupting a surface loop, although there are examples of successful permutation
sites at the former locations [5–10]. The same considerations apply
to the case where the binding mutation is near the N-terminus,
except the duplicated segment begins with the N-terminus and ends
at a surface loop C-terminal to the binding mutation (Fig. 1a).
Inability to find a stable circular permutant (CP) is the most
common failure point in the AFF protocol. Permutation almost
always destabilizes a protein, and there is no reliable method for
predicting the extent of destabilization for a given permutation site.
The CP needs to be at least marginally stable (ΔG unfold ≥ 2–3 kcal/mol),
with more stable CPs requiring less optimization (see Subheading 5).
Our approach for selecting permutation sites is to choose the first
three to four surface loops either N-terminal or C-terminal to the
binding mutation, depending on whether the binding mutation is
closer to the C-terminus or N-terminus, respectively. Loops that are
close to the binding/active site should be avoided for functional
reasons, although xylanase [9] and beta lactamase [11] were permuted at several loops proximal to their active sites without major
loss of activity. Fortunately, all but the smallest POIs will have many
loops from which to choose and at least one will usually be stable
and functional enough for the AFF design. For example, RBP (277
amino acids) has 13 surface loops (Fig. 2a). We created CPs at eight
of these loops and all were stable, soluble, and functional. All were
destabilized compared to wild-type (WT) RBP, however, and this
finding demonstrates the advantage of starting with the most stable
variant of the POI available.
The linker functions to physically bridge the original N- and
C-termini of the POI. It effectively becomes a new surface loop of
the CP. As such, the amino acid sequence should be hydrophilic
and flexible enough to not impose any new constraints on the
protein structure. We base our linkers on Gly/Ala/Ser repeats,
although more advanced design criteria have been discussed
[9, 11–15]. With regard to linker length, a rule of thumb is to
measure the N-to-C distance (C α –C α ) from the structure “as the
3.2 Identify
a Circular Permutation
Site
3.3 Design
the Peptide Linker
Engineering Allosteric Protein Switches