Gene/Protein Disease Symptom Drug Enzyme Compound
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Use of randomized peptide libraries to evolve molecules with new functions provides a means for developing novel regulators of protein activity. Despite the demonstrated power of such approaches for soluble targets, application of this strategy to membrane systems, such as ion channels, remains challenging. Here, we have combined libraries of a tethered protein scaffold with functional selection in yeast to develop a novel activator of the G-protein-coupled mammalian inwardly rectifying potassium channel Kir3.2 (GIRK2). We show that the novel regulator, denoted N5, increases Kir3.2 (GIRK2) basal activity by inhibiting clearance of the channel from the cellular surface rather than affecting the core biophysical properties of the channel. These studies establish the tethered protein display strategy as a means to create new channel modulators and highlight the power of approaches that couple randomized libraries with direct selections for functional effects. Our results further underscore the possibility for the development of modulators that influence channel function by altering cell surface expression densities rather than by direct action on channel biophysical parameters. The use of tethered library selection strategies coupled with functional selection bypasses the need for a purified target and is likely to be applicable to a range of membrane protein systems.
ACS Chem Neurosci 2014 Sep 17
PMID:Tethered protein display identifies a novel Kir3.2 (GIRK2) regulator from protein scaffold libraries. 2502 3

Molybdenum disulfide (MoS2) nanomaterial has recently found various applications in the biomedical field mainly due to its outstanding physicochemical properties. However, little is known about its interactions with biological systems at the atomic level, which intimately relates to the biocompatibility of the material. To provide insights into the effects of MoS2 in biological entities, we investigated the interactions of MoS2 with proteins from a functionally important membrane family, the ubiquitous potassium (K+) channels. Here, we study four representative K+ channels-KcsA, Kir3.2, the Kv1.2 paddle chimera, and K2P2-to investigate their interactions with a triangular MoS2 nanoflake using Molecular Dynamics (MD) simulations combined with electrophysiology experiments. These particular K+ channels were selected based on the diversity in their structure; that is, although these K+ channels display similar structural motifs, they also contain significant differences related to their particular function. Our results indicate that the MoS2 nanoflake is able to stably bind to three out of the four channels, albeit through distinct binding modes. The binding mode between each channel and MoS2 underlies the specific deleterious influence on the channel's basic physiological function: For KcsA, MoS2 binds on the extracellular loops, which indirectly destroys the delicate structure of the selectivity filter causing a strong leak of K+ ions. In the binding mode with Kir3.2, the MoS2 nanoflake completely covers the entrance to the channel pore affecting the normal ion conduction. For the Kv1.2 chimera, the MoS2 nanoflake prefers to bind into a crevice located at the extracellular side of the Voltage Sensor Domain (VSD). Interestingly, the crevice involves the N-terminal segment of S4, a crucial transmembrane helix which directly controls the gating process of the Kv1.2 chimera channel by electromechanical coupling the VSD to the transmembrane electric field. MoS2 in contact with S4 from the Kv1.2 chimera, potentially influences the channel's gating process from open to closed states. In all three systems, the van der Waals contribution to the total energy dominates the binding interactions; also, hydrophobic residues contribute the most contact points, which agrees with the strong hydrophobic character of the MoS2 nanomaterial. Electrophysiology recordings using two-electrode voltage-clamp show that currents of Kir3.2 and Kv1.2 are both blocked by the MoS2 nanoflakes in a concentration-dependent way. While the background K+ channel, K2P2 (TREK-1), identified as a negative control, is not blocked by the MoS2 nanoflakes. The large and rigid extracellular domain of K2P2 appears to protect the channel from disturbance by the nanoflakes. Intrinsic chemical properties of MoS2, together with the specific features of the channels, such as the electrostatic character and complex surface architecture, determine the critical details of the binding events. These findings might shed light on the potential nanotoxicology of MoS2 nanomaterials and help us to understand the underlying molecular mechanism.
ACS Nano 2018 01 23
PMID:Exploring the Nanotoxicology of MoS2: A Study on the Interaction of MoS2 Nanoflakes and K+ Channels. 2923 81