4
featuring allosteric-binding receptors that are inserted into the tertiary structure of an actuator such as a fluorescent protein (FP) or
an autoinhibited enzyme module, to modularly organized binding
receptors and actuators where independently folding functional
domains are organized along a linear polypeptide chain. For integrated designs, a binding event is typically transduced from the
receptor to the actuator through a complex network of conformational transitions in the tertiary structure of a protein. In contrast,
modularly organized synthetic protein switches are typically regulated through mutually exclusive binding interactions where conformational transitions are limited to the linkers connecting
independently folding functional domains. Beyond single-component protein switches, synthetic protein switches can also be composed of multiple molecularly distinct components. These are
typically regulated through the induced proximity of a transducer
with an actuator or two split protein halves [3, 4].
In terms of specific applications, synthetic protein switches are
increasingly employed as intracellular sensors that monitor molecular functions in an integrated and autonomous fashion in real time,
e.g., reporting on the presence or absence of key metabolites, protein-protein interactions, or posttranslational modifications based
on fluorescence or bioluminescence readouts [5–8]. In comparison, conventional techniques that have traditionally been employed
to analyze protein-associated functions by means of antibodies or
mass spectrometry only provide snapshots of molecular states as
cells and tissues need to be broken up and/or fixed for analysis. In
this case, monitoring time courses of biological processes based on
successive time points quickly becomes laborious and also introduces variability from repeated sampling. Beyond applications in
basic research, synthetic protein switches are increasingly developed as diagnostic reagents to detect clinically important biomarkers in an integrated fashion with no need for laborious work-up
steps such as the successive binding and washing steps necessitated
by immunological techniques based on antibodies.
Beyond applications as molecular sensors, synthetic protein
switches can also be employed to actuate biological functions [9–
12]. Traditionally, this has been realized through small molecular
weight ligands that can bind and thus control the function of key
signaling proteins inside the cell. In the majority of cases, small
molecular weight ligands primarily inhibit protein-associated functions. In contrast, synthetic protein switches can regulate cellular
functions in both positive and negative ways, for instance, by
introducing artificial control elements into key regulatory proteins
of intracellular signal transduction pathways.
With a number of applications emerging in basic research
and biotechnology, a key bottleneck has been to devise generally applicable strategies to engineer synthetic protein switches
with tailored response functions [1, 2, 13, 14]. Notably, current
Viktor Stein
featuring allosteric-binding receptors that are inserted into the tertiary structure of an actuator such as a fluorescent protein (FP) or
an autoinhibited enzyme module, to modularly organized binding
receptors and actuators where independently folding functional
domains are organized along a linear polypeptide chain. For integrated designs, a binding event is typically transduced from the
receptor to the actuator through a complex network of conformational transitions in the tertiary structure of a protein. In contrast,
modularly organized synthetic protein switches are typically regulated through mutually exclusive binding interactions where conformational transitions are limited to the linkers connecting
independently folding functional domains. Beyond single-component protein switches, synthetic protein switches can also be composed of multiple molecularly distinct components. These are
typically regulated through the induced proximity of a transducer
with an actuator or two split protein halves [3, 4].
In terms of specific applications, synthetic protein switches are
increasingly employed as intracellular sensors that monitor molecular functions in an integrated and autonomous fashion in real time,
e.g., reporting on the presence or absence of key metabolites, protein-protein interactions, or posttranslational modifications based
on fluorescence or bioluminescence readouts [5–8]. In comparison, conventional techniques that have traditionally been employed
to analyze protein-associated functions by means of antibodies or
mass spectrometry only provide snapshots of molecular states as
cells and tissues need to be broken up and/or fixed for analysis. In
this case, monitoring time courses of biological processes based on
successive time points quickly becomes laborious and also introduces variability from repeated sampling. Beyond applications in
basic research, synthetic protein switches are increasingly developed as diagnostic reagents to detect clinically important biomarkers in an integrated fashion with no need for laborious work-up
steps such as the successive binding and washing steps necessitated
by immunological techniques based on antibodies.
Beyond applications as molecular sensors, synthetic protein
switches can also be employed to actuate biological functions [9–
12]. Traditionally, this has been realized through small molecular
weight ligands that can bind and thus control the function of key
signaling proteins inside the cell. In the majority of cases, small
molecular weight ligands primarily inhibit protein-associated functions. In contrast, synthetic protein switches can regulate cellular
functions in both positive and negative ways, for instance, by
introducing artificial control elements into key regulatory proteins
of intracellular signal transduction pathways.
With a number of applications emerging in basic research
and biotechnology, a key bottleneck has been to devise generally applicable strategies to engineer synthetic protein switches
with tailored response functions [1, 2, 13, 14]. Notably, current
Viktor Stein