Site-Directed Fluorescence Approaches for Dynamic Structural Biology of Membrane Peptides and Proteins

Abstract

Membrane proteins mediate a number of cellular functions and are associated with several diseases and also play a crucial role in pathogenicity. Due to their importance in cellular structure and function, they are important drug targets for ~60% of drugs available in the market. Despite the technological advancement and recent successful outcomes in determining the high-resolution structural snapshot of membrane proteins, the mechanistic details underlining the complex functionalities of membrane proteins is least understood. This is largely due to lack of structural dynamics information pertaining to different functional states of membrane proteins in a membrane environment. Fluorescence spectroscopy is a widely used technique in the analysis of functionally-relevant structure and dynamics of membrane protein. This review is focused on various site-directed fluorescence (SDFL) approaches and their applications to explore structural information, conformational changes, hydration dynamics, and lipid-protein interactions of important classes of membrane proteins that include the pore-forming peptides/proteins, ion channels/transporters and G-protein coupled receptors.

The role of structural dynamics in GPCR‐mediated signaling

“…Therefore, alternative biophysical techniques including nuclear magnetic resonance (NMR) (reviewed in Ref. [58]), electron paramagnetic resonance (EPR) (reviewed in Refs [59,60]), and fluorescence spectroscopy (reviewed in Refs [61,62]) supported by molecular dynamics (MD) simulations (reviewed in Refs [63,64]) have been used in complementation to crystallographic and cryo-EM studies to analyze different conformational states of GPCRs and to determine their liganddependent energetics and rates of interconversion. Several NMR studies on the b 1 and b 2 adrenergic receptors (b 1 AR and b 2 AR) [65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80], the adenosine A 2A receptor (A 2A R) [81][82][83][84][85][86][87], the l-opioid receptor (lOR) [88,89], the leukotriene B 4 receptor 2 (BLT2R) [90], the a 1A adrenergic receptor (a 1A R) [91], the neurotensin receptor type 1 (NTSR1) [92], and the M 2 R [93,94] have shown that GPCRs are highly dynamic proteins that exist in an equilibrium between multiple functionally relevant conformational states.…”

Structural Dynamics of the Paddle Motif Loop in the Activated Conformation of KvAP Voltage Sensor

“…It is well established that NBD is weakly fluorescent in water, fluoresces brightly in the visible range upon transfer to a hydrophobic medium, and exhibits a high degree of environmental sensitivity (32)(33)(34), which has been widely used to monitor the dynamics of membranes (35,37) and membrane proteins (see (25,26) for reviews; (31,(38)(39)(40)). Further, it is known that depending on temperature, the fluorescence emission maximum of NBD is 517-522 and 530-535 nm when placed in the hydrophobic core (35) and in the interfacial region of the membrane (37), respectively.…”

“…1 A) using site-specific labeling with an environment-sensitive fluorophore 7-nitrobenz-2-oxa-1,3diazol-4-yl (NBD) in OG micelles and PC/PG membranes. For this purpose, we have utilized various site-directed fluorescence approaches (25,26) such as emission maximum, lifetime, polarization, red edge excitation shift (REES), and accessibility (quenching) studies in micelles and membranes. Our results show that the S3b-S4 loop residues partition at the membrane interface, and the structural dynamics of this physiologically important S3b-S4 loop is significantly altered in membrane environment.…”

Neuropathy target esterase (NTE/PNPLA6) and organophosphorus compound-induced delayed neurotoxicity (OPIDN)

“…The fact that NTE is a large membrane-bound and multi-domain molecule hinders structural determination by X-ray crystallography. Nevertheless, solving its individual domains can be undertaken using X-ray and NMR techniques (Puthenveetil and Vinogradova, 2019), and determining how its domains interact with each other can be approached by combining site-directed fluorescence, chemical crosslinking, isotope exchange, and mass spectrometry (Hodge et al, 2019;Raghuraman et al, 2019;Xiang et al, 2020). In addition, cryogenic electron microscopy has recently emerged as an ideal technique for solving the structures of "difficult" proteins, and its resolution is now approaching that of X-ray methods (Nwanochie and Uversky, 2019;Schur, 2019).…”