Toll-like receptors (TLRs) are participants of the innate immune system that perceive the presence of pathogens, initiating the inflammation. The key event of their activation is dimerization upon ligand recognition. Signal transduction through the membrane is mediated by transmembrane and juxtamembrane regions. However, nothing is known about the structure of the transmembrane and intracellular parts of the receptors in the activated dimeric state. Here, we investigate the dimerization of transmembrane and juxtamembrane parts (TMJMs) of TLR1 and TLR2 in lipid bilayer-containing particles using NMR spectroscopy. We found that transmembrane domains of TLR1 and TLR2 both homo- and heterodimerize, with heterodimerization being tenfold stronger compared to homotypic interaction. The heterodimer is formed via an extensive interaction interface leading to local changes in the hinge and juxtamembrane region preceding the TIR domain. We believe that TMJMs of TLR1 and TLR2 could facilitate signalosome formation affecting the TIR domain.

Fluorescence-lifetime imaging microscopy (FLIM) is a powerful technique for highly multiplexed imaging in live cells. In this work, we present a genetically encoded FLIM multiplexing platform based on a combination of fluorogen-activating protein FAST and red-shifted fluorogen N871b from the arylidene-imidazolone family. We showed that a series of FAST protein mutants exhibit similar steady-state optical properties in complex with N871b fluorogen but have different fluorescence lifetimes. The similar brightness and binding strength of pairs of these FAST protein variants with N871b allows them to be successfully used for multiplexing up to three intracellular structures of living cells simultaneously.

The palette of the fluorogen-activating protein FAST expanded into the far-red region by the development of a novel fluorogen, HBTR-3,5-DOM. This was achieved through a C=O to C=S substitution in the classic hydroxybenzylidene-rhodanine core, which induced a bathochromic shift of over 100 nm. The complexes of HBTR-3,5-DOM with FAST variants pFAST and F62L are characterized by absorption and emission maxima at 640–650 nm and ~670 nm, respectively, and are found to exhibit distinct fluorescence lifetimes. The fluorogen is successfully applied in genetically encoded live-cell imaging together with these FAST variants for various subcellular structures. Furthermore, its potential for multiplexed imaging is demonstrated by the simultaneous discrimination of two targeted proteins using fluorescence lifetime imaging microscopy (FLIM).

Toll-like receptors (TLRs) are important players in the innate immune system. Binding of pathogen-related molecules to the extracellular domains of TLRs initiates signalosome assembly, a key event in signal transduction. Despite extensive research on individual receptor domains, the mechanism of signalosome assembly remains unclear. Recent evidence suggests that the intracellular TIR domain of TLR1 binds zinc ions, with cysteines playing a pivotal role in binding and receptor activation. This study explores the zinc-binding ability of the TLR2 TIR domain (TLR2TIR). We found that TLR2TIR binds zinc with nanomolar affinity through its cysteine residues. Two of them, C673 and C713, are essential for receptor activation. These results suggest that zinc may be involved in the initiation of signalosome assembly.

Objective: One of the major problems in the field of neurotrophin signaling is the role of Trk juxtamembrane regions. Here we present the production protocol of the d5 ligand-binding domain of TrkA with the full-length extracellular juxtamembrane region for structural studies. Methods: The protein was produced in E. coli cells. Protein purification included immobilized metal affinity and size-exclusion chromatography in the presence of urea. Refolding was performed using three approaches: dialysis, pulse and flash dilution. The quality of the final protein was assessed by gel filtration and NMR. Results and Discussion: We demonstrated that the obtained strain allows the production of milligram quantities of the target protein, including its isotope-labeled derivatives. A comparison of several refolding protocols revealed that dialysis and flash dilution are optimal, with the latter option being more economically feasible. Conclusions: Analysis of the final protein preparation showed that the proposed protein expression, purification, and refolding scheme allows the production of a highly purified protein suitable for structural and functional studies.

In this paper, we propose a fluorescence-lifetime imaging microscopy (FLIM) multiplexing system based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform employs FAST mutants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair. All the proposed probes with varying lifetimes possess nearly identical and the smallest-in-class size, along with quite similar steady-state optical properties. In live mammalian cells, we target these chemogenetic tags to two intracellular structures simultaneously, where their fluorescence signals are clearly distinguished by FLIM. Due to the unique structure of certain fluorogens under study, their complexes with FAST mutants display a monophasic fluorescence decay, which may facilitate enhanced multiplexing efficiency by reducing signal cross-talks and providing optimal prerequisites for signal separation upon co-localized and/or spatially overlapped labeling. A genetically encoded labeling system uses smallest-in-class fluorogen-activating protein tags for time-resolved fluorescence multiplexed cellular imaging, offering monoexponential decay and potential for sophisticated fluorescence lifetime analysis.

NanoFAST is the smallest fluorogen-activating protein, consisting of only 98 amino acids, used as a genetically encoded fluorescent tag. Previously, only a single fluorogen with an orange color was revealed for this protein. In the present paper, using rational mutagenesis and in vitro screening of fluorogens libraries, we expanded the color palette of this tag. We discovered that E46Q is one of the key substitutions enabling the range of possible fluorogens to be expanded. The introduction of this and several other substitutions has made it possible to use not only orange but also red and green fluorogens with the modified protein.

Toll-like receptors (TLRs) are key players in the innate immune system. Despite the great efforts in TLR structural biology, today we know the spatial structures of only four human TLR intracellular TIR domains. All of them belong to one of five subfamilies of receptors. One of the main bottlenecks is the high-level production of correctly folded proteins in soluble form. Here we used a rational approach to find the optimal parameters to produce TIR domains of all ten human TLR family members in soluble form in E. coli cells. We showed that dozens of milligrams of soluble His-tagged TLR2/3/6/7TIR and MBP-tagged TLR3/5/7/8TIR can be produced. We also developed the purification protocols and demonstrated by CD and NMR spectroscopy that purified TLR2/3/7TIR demonstrate a structural organization inherent to TIR domains. This illustrates the correct folding of produced proteins and their suitability for further structural and functional investigations.

Membrane proteins are one of the keystone objects in molecular biology, but their structural studies often require an extensive search for an appropriate membrane-like environment and an efficient refolding protocol for a recombinant protein. Isotropic bicelles are a convenient membrane mimetic used in structural studies of membrane proteins. Helical membrane domains are often transferred into bicelles from trifluoroethanol–water mixtures. However, the protocols for such a refolding are empirical and the process itself is still not understood in detail. In search of the optimal refolding approaches for helical membrane proteins, we studied here how membrane proteins, lipids, and detergents interact with each other at various trifluoroethanol–water ratios. Using high-resolution NMR spectroscopy and dynamic light scattering, we determined the key states of the listed compounds in the trifluoroethanol/water mixture, found the factors that could be critical for the efficiency of refolding, and proposed several most optimal protocols. These protocols were developed on the transmembrane domain of neurotrophin receptor TrkA and tested on two model helical membrane domains—transmembrane of Toll-like receptor TLR9 and voltage-sensing domain of a potassium channel KvAP.

Integral membrane proteins are important components of a cell. Their structural and functional studies require production of milligram amounts of proteins, which nowadays is not a routine process. Cell-free protein synthesis is a prospective approach to resolve this task. However, there are few known membrane mimetics that can be used to synthesize active membrane proteins in high amounts. Here, we present the application of commercially available "Facade" detergents for the production of active rhodopsin. We show that the yield of active protein in lipid bicelles containing Facade-EM, Facade-TEM, and Facade-EPC is several times higher than in the case of conventional bicelles with CHAPS and DHPC and is comparable to the yield in the presence of lipid-protein nanodiscs. Moreover, the effects of the lipid-to-detergent ratio, concentration of detergent in the feeding mixture, and lipid composition of the bicelles on the total, soluble, and active protein yields are discussed. We show that Facade-based bicelles represent a prospective membrane mimetic, available for the production of membrane proteins in a cell-free system.

The biochemical obtaining of natively folded membrane proteins (MPs) is an extremely important task of modern structural biology. We have studied the efficiency of co-translational incorporation of MPs into various membrane-mimicking media — liposomes, micelles, and bicelles during the cell-free synthesis. The retinal protein of Exiguobacterium sibiricum (ESR) was chosen as an object of study.

Toll-like receptors (TLRs) are the important participants of the innate immune response. Their spatial organization is well studied for the ligand-binding domains, while a lot of questions remain unanswered for the membrane and cytoplasmic regions of the proteins. Here we use solution NMR spectroscopy and computer simulations to investigate the spatial structures of transmembrane and cytoplasmic juxtamembrane regions of TLR2, TLR3, TLR5, and TLR9. According to our data, all the proteins reveal the presence of a previously unreported structural element, the cytoplasmic hydrophobic juxtamembrane α-helix. As indicated by the functional tests in living cells and bioinformatic analysis, this helix is important for receptor activation and plays a role, more complicated than a linker, connecting the transmembrane and cytoplasmic parts of the proteins.

NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH–N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein.

“Fluorescence-Activating and absorption-Shifting Tag” (FAST) is a well-studied fluorogen-activating protein with high brightness and low size, able to activate a wide range of fluorogens. This makes FAST a promising target for both protein and fluorogen optimization. Here, we describe the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen. Using the spatial structure of the FAST/ N871b complex, NMR relaxation analysis, and computer simulations, we identify the mobile regions in the complex and suggest mutations that could stabilize both the protein and the ligand. Two of our mutants appear brighter than the wild-type FAST, and these mutants provide up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo. Analysis of the mutants by NMR reveals that brighter mutants demonstrate the highest stability and lowest length of intermolecular H-bonds. Computer simulations provide the structural basis for such stabilization.

T7 expression system is an extremely popular approach for the recombinant protein production in Escherichia coli for structural and functional studies and therapeutic applications. There are many useful tools and successful techniques that allow expressing the desired protein in this system. However, high yield of soluble protein often requires a systematic optimization of a wide range of cell cultivation parameters. Here we analyze the effect of three key cultivation parameters - chemical inductor, temperature and time of post-induction culturing on the expression level of TLR1 intracellular TIR domain in a soluble form. In addition, the influence of Triton X-100 detergent on the protein solubility during the cell lysis was investigated. We show that a high expression level of the correctly folded soluble protein can be obtained under different combinations of cultivation parameters.