Development Of Chemical Tools For Biological Studies Of Integral Membrane Proteins

Integral membrane proteins,which represent the main membrane protein?MP?superfamily,play a vital role in many cell physiological activities,such as signal transduction,material exchange and energy transfer of cells.Many diseases are closely related to the dysregulation of signaling pathways caused by abnormal membrane protein functions.Appropriate chemical tools are of great significance for obtaining structural information of membrane proteins,elucidating the signaling mechanism of cells,and promoting the development of new drugs.This dissertation aims to provide important tools for biological research of integrated membrane proteins and consist of the following three parts:Part ?:Owing to stringent requirements of integral MP manipulations,amphiphile replacement is often required in sample preparation for various biophysical purposes.However,current physical protocols rarely reach complete replacement.We reported herein a set of chemically cleavable detergent?CCDs?facilitating an alternative chemical strategy for amphiphile replacement.In this context,CCD-2 was proven to not only have excellent solubilization efficiency and comparable stabilization relative to the conventional detergent,but also readily decompose under mild conditions.Therefore,a facile,efficient and traceless chemical method was then developed to exhaustively in situ shift the MP shield from the CCD micelles to other amphiphile cassettes facilitating various biophysical characterization.Based on this novel strategy,the A_2A adenosine receptor was successfully reconstituted into a series of commercial detergents to guide the selection of proper amphiphile candidates for maintenance of MPs in native monotopic state.Meanwhile,this strategy also found to be amenable to reconstituting MPs into highly homogeneous nanodiscs,from which decent negative staining EM data were collected for future structural elucidation.Part ?:Detergents are indispensable membrane tools in the structural and functional understanding of MPs.Given that fragile MPs that are reconstituted in conventional detergents tend to either lose their native-like states or manifest unfavorable properties for structural and functional studies,there is still a need for new,well-tuned detergents.We presented here a new class of disulfide-containing detergents?DCDs?for structural investigations of MPs using NMR technology.Thanks to the distinct nature of disulfide,all DCDs were found to form smaller micelles than their counterpart DDM.Representative DCDs behaved well in the refolding of the?-barrel protein OmpX,and in the solubilization of G protein-coupled receptors?GPCRs?.The characteristics of DCD's yield outstanding performance in solution NMR studies of MPs,as demonstrated by the collection and assignment of 2D[¹⁵N,¹H]-TROSY spectra of OmpX,and by 1D ¹⁹F NMR and 2D[¹⁵N,¹H]-TROSY spectra of A_2AAR.These above advantages offered by these DCDs over its counterpart indicate this alternative design strategy for NMR studies of MPs is an important step for future detergent development.Part ?:Smoothened receptor?SMO?is a class F GPCR and plays a crucial role in embryonic development and tumorigenesis.Visualization of SMO bears the potential to prompt new insights into its enigmatic signal transmission mechanisms and associated disease pathogenesis.Based on recent progress in structural studies of SMO,we designed and characterized a group of affinity probes facilitating turn-on fluorescence labeling SMO.These chemical probes were derived from a potent SMO antagonist skeleton by conjugation of a non-fluorescent unit O-nitrobenzoxadiazole?O-NBD?.In this context,the optimal probes were evolved to be capable of efficiently and selectively lighting up SMO at the?-amine of K395,regardless of whether it is in micelles or in native membranes.Notably,the resulting labeled SMO only bears a very small fluorophore and allows for recovery of unoccupied pocket by dissociation of the residual ligand part.These advantages should promise this probe to serve as a potential tool for monitoring SMO trafficking,understanding Hedgehog activation mechanisms,and even diagnosis of tumorigenesis in the future.