Under anaerobic conditions, bacteria may utilize nitrates and nitrites as electron acceptors. Sensitivity to nitrous compounds is achieved via several mechanisms, some of which rely on sensor ...histidine kinases (HKs). The best studied nitrate- and nitrite-sensing HKs (NSHKs) are NarQ and NarX from Escherichia coli. Here, we review the function of NSHKs, analyze their natural diversity, and describe the available structural information. In particular, we show that around 6000 different NSHK sequences forming several distinct clusters may now be found in genomic databases, comprising mostly the genes from Beta- and Gammaproteobacteria as well as from Bacteroidetes and Chloroflexi, including those from anaerobic ammonia oxidation (annamox) communities. We show that the architecture of NSHKs is mostly conserved, although proteins from Bacteroidetes lack the HAMP and GAF-like domains yet sometimes have PAS. We reconcile the variation of NSHK sequences with atomistic models and pinpoint the structural elements important for signal transduction from the sensor domain to the catalytic module over the transmembrane and cytoplasmic regions spanning more than 200 Å.
Here, we propose a possible photoactivation mechanism of a 35-kDa blue light-triggered photoreceptor, the Orange Carotenoid Protein (OCP), suggesting that the reaction involves the transient ...formation of a protonated ketocarotenoid (oxocarbenium cation) state. Taking advantage of engineering an OCP variant carrying the Y201W mutation, which shows superior spectroscopic and structural properties, it is shown that the presence of Trp201 augments the impact of one critical H-bond between the ketocarotenoid and the protein. This confers an unprecedented homogeneity of the dark-adapted OCP state and substantially increases the yield of the excited photoproduct S*, which is important for the productive photocycle to proceed. A 1.37 Å crystal structure of OCP Y201W combined with femtosecond time-resolved absorption spectroscopy, kinetic analysis, and deconvolution of the spectral intermediates, as well as extensive quantum chemical calculations incorporating the effect of the local electric field, highlighted the role of charge-transfer states during OCP photoconversion.
Glucose-methanol-choline (GMC) oxidoreductases are a large and diverse family of flavin-binding enzymes found in all kingdoms of life. Recently, a new related family of proteins has been discovered ...in algae named fatty acid photodecarboxylases (FAPs). These enzymes use the energy of light to convert fatty acids to the corresponding Cn-1 alkanes or alkenes, and hold great potential for biotechnological application. In this work, we aimed at uncovering the natural diversity of FAPs and their relations with other GMC oxidoreductases. We reviewed the available GMC structures, assembled a large dataset of GMC sequences, and found that one active site amino acid, a histidine, is extremely well conserved among the GMC proteins but not among FAPs, where it is replaced with alanine. Using this criterion, we found several new potential FAP genes, both in genomic and metagenomic databases, and showed that related bacterial, archaeal and fungal genes are unlikely to be FAPs. We also identified several uncharacterized clusters of GMC-like proteins as well as subfamilies of proteins that lack the conserved histidine but are not FAPs. Finally, the analysis of the collected dataset of potential photodecarboxylase sequences revealed the key active site residues that are strictly conserved, whereas other residues in the vicinity of the flavin adenine dinucleotide (FAD) cofactor and in the fatty acid-binding pocket are more variable. The identified variants may have different FAP activity and selectivity and consequently may prove useful for new biotechnological applications, thereby fostering the transition from a fossil carbon-based economy to a bio-economy by enabling the sustainable production of hydrocarbon fuels.
Light-oxygen-voltage (LOV) domains are ubiquitous photosensory modules found in proteins from bacteria, archaea and eukaryotes. Engineered versions of LOV domains have found widespread use in ...fluorescence microscopy and optogenetics, with improved versions being continuously developed. Many of the engineering efforts focused on the thermal stabilization of LOV domains. Recently, we described a naturally thermostable LOV domain from Chloroflexus aggregans. Here we show that the discovered protein can be further stabilized using proline substitution. We tested the effects of three mutations, and found that the melting temperature of the A95P mutant is raised by approximately 2 °C, whereas mutations A56P and A58P are neutral. To further evaluate the effects of mutations, we crystallized the variants A56P and A95P, while the variant A58P did not crystallize. The obtained crystal structures do not reveal any alterations in the proteins other than the introduced mutations. Molecular dynamics simulations showed that mutation A58P alters the structure of the respective loop (Aβ-Bβ), but does not change the general structure of the protein. We conclude that proline substitution is a viable strategy for the stabilization of the Chloroflexus aggregans LOV domain. Since the sequences and structures of the LOV domains are overall well-conserved, the effects of the reported mutations may be transferable to other proteins belonging to this family.
TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in ...inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.
Further insight into the structural biology of TRP channels is crucial to explain molecular mechanisms of channel function.
We purified TRPV3, demonstrated its functional integrity, and used fluorescence spectroscopy to study ligand binding.
TRPV3 ligands induce different conformational changes as observed by tryptophan fluorescence quenching.
Availability of purified TRPV3 allows functional assays outside the cellular context and facilitates future structural studies.
Amphiphilic copolymers consisting of alternating hydrophilic and hydrophobic units account for a major recent methodical breakthrough in the investigations of membrane proteins. Styrene-maleic acid ...(SMA), diisobutylene-maleic acid (DIBMA), and related copolymers have been shown to extract membrane proteins directly from lipid membranes without the need for classical detergents. Within the particular experimental setup, they form disc-shaped nanoparticles with a narrow size distribution, which serve as a suitable platform for diverse kinds of spectroscopy and other biophysical techniques that require relatively small, homogeneous, water-soluble particles of separate membrane proteins in their native lipid environment. In recent years, copolymer-encased nanolipoparticles have been proven as suitable protein carriers for various structural biology applications, including cryo-electron microscopy (cryo-EM), small-angle scattering, and conventional and single-molecule X-ray diffraction experiments. Here, we review the current understanding of how such nanolipoparticles are formed and organized at the molecular level with an emphasis on their chemical diversity and factors affecting their size and solubilization efficiency.
Histidine kinases constitute an abundant class of membrane receptors present in all domains of life. Many of them are essential for cell growth, survival, or pathogenicity. However, the mechanisms of ...transmembrane signaling by such sensors are not fully understood due to the scarcity of structural information on full‐length proteins. We used styrene–maleic acid (SMA) copolymers to solubilize full‐length nitrate/nitrite sensor histidine kinase NarQ from Escherichia coli. The resulting SMA lipid particles (SMALPs) provided a native membrane‐like environment for NarQ and remained stable for an extended period of time. We characterized NarQ‐containing SMALPs using size‐exclusion chromatography and small angle X‐ray scattering. Overall, the particles were heterogeneous in size, with the scattering from the smallest ones matching the theoretical scattering from a single NarQ dimer embedded in a SMALP comprising ~150 SMA molecules and ~350 lipids. These results show that SMA can be used to extract full‐length histidine kinases from the membrane with efficiency comparable to, and exceeding that of the commonly used detergent dodecyl maltoside (DDM), and the resulting samples are suitable for structural studies.
Recent advances in machine learning techniques have led to development of a number of protein design and engineering approaches. One of them, ProteinMPNN, predicts an amino acid sequence that would ...fold and match user-defined backbone structure. Its performance was previously tested for proteins composed of standard amino acids, as well as for peptide- and protein-binding proteins. In this short report, we test whether ProteinMPNN can be used to reengineer a non-proteinaceous ligand-binding protein, flavin-based fluorescent protein CagFbFP. We fixed the native backbone conformation and the identity of 20 amino acids interacting with the chromophore (flavin mononucleotide, FMN) while letting ProteinMPNN predict the rest of the sequence. The software package suggested replacing 36-48 out of the remaining 86 amino acids so that the resulting sequences are 55%-66% identical to the original one. The three designs that we tested experimentally displayed different expression levels, yet all were able to bind FMN and displayed fluorescence, thermal stability, and other properties similar to those of CagFbFP. Our results demonstrate that ProteinMPNN can be used to generate diverging unnatural variants of fluorescent proteins, and, more generally, to reengineer proteins without losing their ligand-binding capabilities.
Light-oxygen-voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the ...currently known LOV domains have absorption maxima in the range of ~440 to ~450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue-shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced byacidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 Å for Q148D to 1.63 Å for Q148R. Whereas in some of the variants, the aminoacid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C-terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged aminoacids and pave the way for rational design of color-shifted flavin based fluorescent proteins.
LOV domains are widespread photosensory modules that have also found applications in fluorescence microscopy, optogenetics, and light-driven generation of reactive oxygen species. Many of these ...applications require stable proteins with altered spectra. Here, we report a flavin-based fluorescent protein CisFbFP derived from Chloroflexus islandicus LOV domain-containing protein. We show that CisFbFP is thermostable, and its absorption and fluorescence spectra are red-shifted for ∼6 nm, which has not been observed for other cysteine-substituted natural LOV domains. We also provide a crystallographic structure of CisFbFP at the resolution of 1.2 Å that reveals alterations in the active site due to replacement of conservative asparagine with a serine. Finally, we discuss the possible effects of presence of cis-proline in the Aβ-Bβ loop on the protein's structure and stability. The findings provide the basis for engineering and color tuning of LOV-based tools for molecular biology.
•Chloroflexus islandicus genome encodes a red-shifted LOV domain.•Homologous mutation results in red shift of fluorescence spectra in YtvA but not in CagFbFP.•High-resolution crystal structure reveals altered active site and additional water molecule.•Presence of cis-proline in the Aβ-Bβ loop changes its structure in CisFbFP.
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