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1 Introduction
A biosensor is minimally composed of an input module, which
interacts with the analyte, and an output module that reports on
that interaction. Proteins excel at both roles. As input domains
they are masters of molecular recognition, having the ability to
bind targets tightly and specifically amidst a sea of similar-looking
decoys. As output domains they possess a wide array of biological
functions, among the most useful of which for biosensing are
fluorescence and enzymatic activity. A major additional advantage
of a protein-based biosensor is that it is genetically encodable for
in vivo applications.
The main challenge in designing a protein-based biosensor is
solving the problem of how to couple input and output domains,
both physically and functionally, so that binding the analyte produces a detectable signal. Nature has given us some treasured, but
rare clues in the form of proteins that undergo large-scale conformational changes in response to ligand binding (e.g., calmodulin,
which has launched a family of fluorescent calcium sensors). The
great majority of proteins, however, do not change their structure
appreciably upon binding.
Calbindin D 9k , fibronectin 3 (Fn3), and ribose binding protein
(RBP) are three such examples in which the structures of the proteins in their ligand-free and ligand-bound states are similar. To
address this challenge, we developed two methodologies for engineering a large, binding-dependent conformational change into
each protein, which was then detected by placement of either
chemical fluorophores or fluorescent proteins (FPs) [1–4]. The
methods are known as alternate frame folding (AFF) and protein/
fragment exchange (FREX).
AFF and FREX both use partial sequence duplication to give a
protein of interest (POI) a mutually exclusive choice between folding to its normal native state (N) or to an alternate form (N′) that
possesses native-like structure and function (Fig. 1). Step 1 (see
Subheading 3) of the protocol for creating an AFF-modified POI
(POI-AFF) involves choosing an appropriate N-terminal or
C-terminal segment of the POI to duplicate. One or more amino
acids are identified in the segment that, when mutated, abrogate
binding of the POI to its target ligand. Step 2 (see Subheading 4)
entails attaching the duplicate copy of the N-terminal or C-terminal
segment to the C- or N-terminus of the POI, respectively, by
means of a peptide linker. Fig. 1a illustrates that POI-AFF can
either fold by using the normal order of amino acids to yield N, or
by using a rearranged order of amino acids to generate a circularly
permuted structure (N′). The relative thermodynamic stabilities of
N and N′ are tuned in Step 3 (see Subheading 5) of the protocol
such that POI-AFF is predominantly in state N (or N′) in the
Jeung-Hoi Ha and Stewart N. Loh
1 Introduction
A biosensor is minimally composed of an input module, which
interacts with the analyte, and an output module that reports on
that interaction. Proteins excel at both roles. As input domains
they are masters of molecular recognition, having the ability to
bind targets tightly and specifically amidst a sea of similar-looking
decoys. As output domains they possess a wide array of biological
functions, among the most useful of which for biosensing are
fluorescence and enzymatic activity. A major additional advantage
of a protein-based biosensor is that it is genetically encodable for
in vivo applications.
The main challenge in designing a protein-based biosensor is
solving the problem of how to couple input and output domains,
both physically and functionally, so that binding the analyte produces a detectable signal. Nature has given us some treasured, but
rare clues in the form of proteins that undergo large-scale conformational changes in response to ligand binding (e.g., calmodulin,
which has launched a family of fluorescent calcium sensors). The
great majority of proteins, however, do not change their structure
appreciably upon binding.
Calbindin D 9k , fibronectin 3 (Fn3), and ribose binding protein
(RBP) are three such examples in which the structures of the proteins in their ligand-free and ligand-bound states are similar. To
address this challenge, we developed two methodologies for engineering a large, binding-dependent conformational change into
each protein, which was then detected by placement of either
chemical fluorophores or fluorescent proteins (FPs) [1–4]. The
methods are known as alternate frame folding (AFF) and protein/
fragment exchange (FREX).
AFF and FREX both use partial sequence duplication to give a
protein of interest (POI) a mutually exclusive choice between folding to its normal native state (N) or to an alternate form (N′) that
possesses native-like structure and function (Fig. 1). Step 1 (see
Subheading 3) of the protocol for creating an AFF-modified POI
(POI-AFF) involves choosing an appropriate N-terminal or
C-terminal segment of the POI to duplicate. One or more amino
acids are identified in the segment that, when mutated, abrogate
binding of the POI to its target ligand. Step 2 (see Subheading 4)
entails attaching the duplicate copy of the N-terminal or C-terminal
segment to the C- or N-terminus of the POI, respectively, by
means of a peptide linker. Fig. 1a illustrates that POI-AFF can
either fold by using the normal order of amino acids to yield N, or
by using a rearranged order of amino acids to generate a circularly
permuted structure (N′). The relative thermodynamic stabilities of
N and N′ are tuned in Step 3 (see Subheading 5) of the protocol
such that POI-AFF is predominantly in state N (or N′) in the
Jeung-Hoi Ha and Stewart N. Loh