Triglyceride Assay Archives - Gen9 Genetics
Allostery is a fundamental protein regulation principle that remains difficult to engineer, particularly in membrane proteins such as ion channels. Here we use the human Kir2.1 K + Channel Internal Rectifier to map site-specific allowability to insertion of domains with different biophysical properties. We found that permissibility is best explained by the dynamic properties of proteins, such as conformational flexibility. Several regions in Kir2.1 that are equivalent to homologous-regulated ones, such as G-protein-activated internal rectifier K + channels (GIRK), have differential permissibility; that is, for these sites the permissibility depends on the structural properties of the inserted domain. Our data and the well-established link between protein dynamics and allosteria led us to propose that differential permissibility is a metric of latent allosteric capacity in Kir2.1. In support of this notion, the insertion of light-switchable domains at sites with predicted latent allosteric capacity renders Kir2.1 activity sensitive to light.
Allostery is the phenomenon in proteins where the state of the proximal sites is coupled to the state of the distal sites. In nature, allosteric regulation is widespread in multidomain proteins, such as plant photoreceptors1, which arise from the recombination of functionally and structurally discrete protein domains. In the laboratory, we recombine domains to generate synthetic proteins; for example, antibodies that bind end-to-end with signaling domains to create chimeric T cell receptors for immunotherapy2. In both scenarios, the way these components are allosterically coupled is essentially trial and error. The fact that blind trial and error can progressively lead to optimized design through natural selection over billions of years is a central concept in the evolution of natural systems3. However, in the laboratory, we need to do this in less time and with greater efficiency.
One class of proteins that are difficult to rationally design are ion channels. Ion channels play a fundamental role in the biological signaling processes that determine the functioning of cells and networks of the brain and heart and, therefore, are the main targets of drugs4. Virtually all aspects of ion channel activation are based on allosteric regulation and many drugs achieve their therapeutic effect through allosteric modulation5. Being able to design the de novo allosteric regulation of ion channels, for example, as chemo or optogenetic tools6,7, would allow precise control and therefore exploration of how individual channels contribute to cell physiology.