Abstract:The superfamily of G protein-coupled receptors (GPCRs) mediates a wide range of physiological responses and serves as an important category of drug targets. Earlier biochemical and biophysical studies have shown that GPCRs exist temporally in an ensemble of interchanging conformations. Single-molecule techniques are ideally suited to understand the dynamic signaling and conformational complexity of G protein-coupled receptors (GPCRs). Here, we review the progress in single-molecule studies on GPCRs. We introdu… Show more
“…There is also active debate regarding the oligomeric state of many GPCRs and how this influences their function. These topics are being actively investigated using single-molecule and super-resolution approaches, and various labeling methods for GPCRs have been extensively reviewed 73 and include incorporation of unnatural amino acids into proteins, fluorescent labeling of GPCR ligands, and the use of nanobodies for labeling GPCRs.…”
Section: Super-resolution Investigations Of Biological Membranesmentioning
Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.
“…There is also active debate regarding the oligomeric state of many GPCRs and how this influences their function. These topics are being actively investigated using single-molecule and super-resolution approaches, and various labeling methods for GPCRs have been extensively reviewed 73 and include incorporation of unnatural amino acids into proteins, fluorescent labeling of GPCR ligands, and the use of nanobodies for labeling GPCRs.…”
Section: Super-resolution Investigations Of Biological Membranesmentioning
Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.
“…The experiments were performed in the presence of 20 mM cysteamine, and 0.01% Tween20 in PBS buffer (pH 7.0) with 0 M NaCl to provide similar conditions to those used for MD simulations 41 . Addition of 0.01% Tween20 prevents non-specific adsorption of peptides to coverslips and related FRET artefacts 51 . ALEX interleaves the “normal” donor-selective excitation with direct excitation of the acceptor and allows the categorization of fluorescence intensity bursts as donor-only, acceptor-only, or smFRET bursts.Figure 6Single-molecule burst analysis of FlAsH-FCMybbR-CoA-AF647.…”
In recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra, correlation spectroscopy and anisotropy decay were used to characterize labelling and to obtain photophysical parameters of free and peptide-bound FlAsH. The data demonstrates that FlAsH is a viable probe for single-molecule studies. Single-molecule imaging confirmed dual labeling of the peptide with FlAsH and AF647. Multiparameter single-molecule Förster Resonance Energy Transfer (smFRET) measurements were performed on freely diffusing peptides in solution. The smFRET histogram showed different peaks corresponding to different backbone and dye orientations, in agreement with the molecular dynamics simulations. The tandem of fluorophores and the labelling strategy described here are a promising alternative to bulky fusion fluorescent proteins for smFRET and single-molecule tracking studies of membrane proteins.
“…However, protein functionalization using enzymatic conjugations is a promising method because it is achieved simply by mixing proteins without special techniques. The details of enzymatic conjugation technology applications will not be covered in this review; readers are referred to several recently published reviews [229–232]. …”
Section: Biomolecular Engineering For Nanobio/bionanotechnologymentioning
confidence: 99%
“…d The TMP-tag noncovalently binds with trimethoprim and brings the α, β-unsaturated carbonyl ( i ) or sulfonyl ( ii ) into proximity of the engineered reactive Cys (L28C)(Figure adapted with permission from: Ref. [229]. Copyright (2017) American Chemical Society)
…”
Section: Biomolecular Engineering For Nanobio/bionanotechnologymentioning
Biomolecular engineering can be used to purposefully manipulate biomolecules, such as peptides, proteins, nucleic acids and lipids, within the framework of the relations among their structures, functions and properties, as well as their applicability to such areas as developing novel biomaterials, biosensing, bioimaging, and clinical diagnostics and therapeutics. Nanotechnology can also be used to design and tune the sizes, shapes, properties and functionality of nanomaterials. As such, there are considerable overlaps between nanotechnology and biomolecular engineering, in that both are concerned with the structure and behavior of materials on the nanometer scale or smaller. Therefore, in combination with nanotechnology, biomolecular engineering is expected to open up new fields of nanobio/bionanotechnology and to contribute to the development of novel nanobiomaterials, nanobiodevices and nanobiosystems. This review highlights recent studies using engineered biological molecules (e.g., oligonucleotides, peptides, proteins, enzymes, polysaccharides, lipids, biological cofactors and ligands) combined with functional nanomaterials in nanobio/bionanotechnology applications, including therapeutics, diagnostics, biosensing, bioanalysis and biocatalysts. Furthermore, this review focuses on five areas of recent advances in biomolecular engineering: (a) nucleic acid engineering, (b) gene engineering, (c) protein engineering, (d) chemical and enzymatic conjugation technologies, and (e) linker engineering. Precisely engineered nanobiomaterials, nanobiodevices and nanobiosystems are anticipated to emerge as next-generation platforms for bioelectronics, biosensors, biocatalysts, molecular imaging modalities, biological actuators, and biomedical applications.
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