Heavy Atom Detergent/Lipid Combined X-ray Crystallography for Elucidating the Structure-Function Relationships of Membrane Proteins
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bacteriorhodopsin receptor↗
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Native Top-Down Mass Spectrometry Characterization of Model Integral Membrane Protein Bacteriorhodopsin
Bacteriorhodopsin (bR) from Halobacterium salinarum has been a model system for structural biology and is a structural template for the characterization of membrane G-protein couple receptors (GPCRs) in particular. Here, in this study, wild-type bacteriorhodopsin and two single-residue mutants were characterized by native top-down mass spectrometry (nTD-MS) with Orbitrap-based high-energy collision dissociation (HCD) and electron capture dissociation (ECD). After in-source dissociation ejected the membrane protein from detergent micelles, high-resolution native MS measurement allowed for identification of multiple proteoforms as well as lipid-bound forms. Further top-down MS measurements by HCD produced a large number of product ions for in-depth sequencing and unambiguous localization of post-translational modifications. For the first time, native TD-MS with ECD was used to characterize an integral membrane protein. ECD yielded fragments originating from all helices and loop regions, even accessing a sequence stretch that HCD could not. Combining HCD and ECD fragmentation patterns significantly enhanced the sequence coverage of bR. We propose bR to be a model analyte for testing nTD-MS performance for membrane proteins.
crystal cleavage↗
Biophysical Characterization of Membrane Proteins Embedded in Nanodiscs Using Fluorescence Correlation Spectroscopy
Proteins embedded in biological membranes perform essential functions in all organisms, serving as receptors, transporters, channels, cell adhesion molecules, and other supporting cellular roles. These membrane proteins comprise ~30% of all human proteins and are the targets of ~60% of FDA-approved drugs, yet their extensive characterization using established biochemical and biophysical methods has continued to be elusive due to challenges associated with the purification of these insoluble proteins. In response, the development of nanodisc techniques, such as nanolipoprotein particles (NLPs) and styrene maleic acid polymers (SMALPs), allowed membrane proteins to be expressed and isolated in solution as part of lipid bilayer rafts with defined, consistent nanometer sizes and compositions, thus enabling solution-based measurements. Fluorescence correlation spectroscopy (FCS) is a relatively simple yet powerful optical microscopy-based technique that yields quantitative biophysical information, such as diffusion kinetics and concentrations, about individual or interacting species in solution. Here, we first summarize current nanodisc techniques and FCS fundamentals. We then provide a focused review of studies that employed FCS in combination with nanodisc technology to investigate a handful of membrane proteins, including bacteriorhodopsin, bacterial division protein ZipA, bacterial membrane insertases SecYEG and YidC, Yersinia pestis type III secretion protein YopB, yeast cell wall stress sensor Wsc1, epidermal growth factor receptor (EGFR), ABC transporters, and several G protein-coupled receptors (GPCRs).
59 BASIC BIOLOGICAL SCIENCES↗
The rhodopsin-like pigments of halobacteria - Light-energy and signal transducers in an archaebacterium
Three, small retinylidene proteins observed in halobacteria are described. The characteristics of bacteriorhodopsin (bR), which is synthesized during low O2 tension and intense illumination, and the role of bR in the cyclic photoreactions that translocate protons are examined. The detected light-driven chloride influx pigment, halorhodopsin (hR), is also capable of light-driven ion translocation; the hR transport reactions which are chloride dependent and involve isomerization are studied. The sensory photosystem of halobacteria and the receptor functions of the retinal pigment slow rhodopsin are discussed. The similarity of the choromphore structure and photoreactions, and the evolutionary relation between halobacteria and animal pigments are considered.
Stoeckenius, W.↗