Powdered G-protein Coupled Receptors

Inventors at the University of Arizona have generated G-protein coupled receptors (GPCRs) lypophilized (freeze-dried) into powdered form. When thawed and rehydrated, these GPCRs retain the functionality of their pre-frozen state even after one year. Because this process also involves the removal of water, the inventors were able to produce thawed GPCRs with specific hydration levels. This appears to be a useful preservation method for GPCRs as well as a way to generate GPCRs with specific concentrations of water (or, as the inventors demonstrated, deuterium) for research purposes.

Background:
This protocol was written as part of an experiment conducted to elucidate the manner by which rhodopsin (a kind of GPCR) mobility is regulated by retinal cofactor of rhodopsin. The nature of the experiment required the substitution of protons with deuterons. These modified GPCRs were produced by multiple freezing/rehydration steps, eventually resulting in the final dehydrated and fully deuterated product. The inventors demonstrated that the powdered GPCRs retained their functionality and structure when rehydrated.

Applications:

• Preservation of GPCRs for long-term storage/shipping. GPCRs processed in this manner retain function for one year following flash-freezing.
• Regulation of water content of GPCRs. GPCRs can be rehydrated to a specific concentration. For experiments that require GPCRs of specific hydration levels (such as the technique used by the inventors, quasi-elastic neutron scattering), this is useful.
• Replacement of water with deuterium in GPCRs. Some experimental techniques require hydrogen to be replaced with deuterons. This technique was demonstrated by the inventors using the powdered GPCRs.

Advantages:

• Centralized manufacture. Increased GPCR stability relative to other processing methods may allow for centralized manufacture and shipping, allowing researchers to bypass the lengthy cloning and purification processes involved in generating GPCRs.
• Lengthier preservation period. Because this method has been shown to preserve GPCR functionality for long periods of time, it may be feasible to generate a large volume of GPCRs with greater efficiency. They could then be thawed as needed with no loss of function.
• Alternative storage mechanism (i.e. as solids). Other GPCR-stabilizing processes are more concerned with how to preserve the GPCR once it has been dissolved in detergents. The method described by the inventors requires minimal detergents and does not require them to remain in liquid solutions.
• Simpler GPCR-stabilizing process. The method described by the inventors does not require the addition of antagonist/agonists (added to force the GPCR into a more stable inactive state) or the addition of any stabilizing lipid-like substances, nor does it require mutating the GPCR to generate a more stable structure. This simplicity may yield stable, unmodified GPCRs more cheaply than other methods.

Status: issued U.S. patents #11,104,717 and #10,526,395.