RESUMO
Light, oxygen, voltage (LOV) photoreceptors are widely distributed throughout all kingdoms of life, and have in recent years, due to their modular nature, been broadly used as sensor domains for the construction of optogenetic tools. For understanding photoreceptor function as well as for optogenetic tool design and fine-tuning, a detailed knowledge of the photophysics, photochemistry, and structural changes underlying the LOV signaling paradigm is instrumental. Mutations that alter the lifetime of the photo-adduct signaling state represent a convenient handle to tune LOV sensor on/off kinetics and, thus, steady-state on/off equilibria of the photoreceptor (or optogenetic switch). Such mutations, however, should ideally only influence sensor kinetics, while being benign with regard to the nature of the structural changes that are induced by illumination, i.e., they should not result in a disruption of signal transduction. In the present study, we identify a conserved hydrophobic pocket for which mutations have a strong impact on the adduct-state lifetime across different LOV photoreceptor families. Using the slow cycling bacterial short LOV photoreceptor PpSB1-LOV, we show that the I48T mutation within this pocket, which accelerates adduct rupture, is otherwise structurally and mechanistically benign, i.e., light-induced structural changes, as probed by NMR spectroscopy and X-ray crystallography, are not altered in the variant. Additional mutations within the pocket of PpSB1-LOV and the introduction of homologous mutations in the LOV photoreceptor YtvA of Bacillus subtilis and the Avena sativa LOV2 domain result in similarly altered kinetics. Given the conserved nature of the corresponding structural region, the here identified mutations should find application in dark-recovery tuning of optogenetic tools and LOV photoreceptors, alike.
Assuntos
Fotorreceptores Microbianos , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/química , Oxigênio/química , Fotoquímica , Mutação , Espectroscopia de Ressonância Magnética , LuzRESUMO
Photoactive biological systems modify the optical properties of their chromophores, known as spectral tuning. Determining the molecular origin of spectral tuning is instrumental for understanding the function and developing applications of these biomolecules. Spectral tuning in flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, is limited by their dependency on protein-bound flavins, whose structure and hence electronic properties cannot be altered by mutation. A blue-shifted variant of the plant-derived improved light, oxygen, voltage FbFP has been created by introducing a lysine within the flavin-binding pocket, but the molecular basis of this shift remains unconfirmed. We here structurally characterize the blue-shifted improved light, oxygen, voltage variant and construct a new blue-shifted CagFbFP protein by introducing an analogous mutation. X-ray structures of both proteins reveal displacement of the lysine away from the chromophore and opening up of the structure as instrumental for the blue shift. Site saturation mutagenesis and high-throughput screening yielded a red-shifted variant, and structural analysis revealed that the lysine side chain of the blue-shifted variant is stabilized close to the flavin by a secondary mutation, accounting for the red shift. Thus, a single additional mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography, and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools for application in life sciences.
Assuntos
Proteínas de Bactérias/química , Chloroflexus/metabolismo , Flavinas/metabolismo , Proteínas Luminescentes/química , Proteínas Mutantes/química , Mutação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chloroflexus/crescimento & desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Simulação de Dinâmica Molecular , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fotoquímica , Conformação Proteica , Teoria QuânticaRESUMO
Light, oxygen, voltage (LOV) proteins, a ubiquitously distributed class of photoreceptors, regulate a wide variety of light-dependent physiological responses. Because of their modular architecture, LOV domains, i.e., the sensory domains of LOV photoreceptors, have been widely used for the construction of optogenetic tools. We recently described the structure and function of a short LOV protein (DsLOV) from the marine phototropic bacterium Dinoroseobacter shibae, for which, in contrast to other LOV photoreceptors, the dark state represents the physiologically relevant signaling state. Among bacterial LOV photoreceptors, DsLOV possesses an exceptionally fast dark recovery, corroborating its function as a "dark" sensor. To address the mechanistic basis of this unusual characteristic, we performed a comprehensive mutational, kinetic, thermodynamic, and structural characterization of DsLOV. The mechanistic basis of the fast dark recovery of the protein was revealed by mutation of the previously noted uncommon residue substitution at position 49 found in DsLOV. The substitution of M49 with different residues that are naturally conserved in LOV domains tuned the dark-recovery time of DsLOV over 3 orders of magnitude, without grossly affecting its overall structure or the light-dependent structural change observed for the wild-type protein. Our study thus provides a striking example of how nature can achieve LOV photocycle tuning by subtle structural alterations in the LOV domain active site, highlighting the easy evolutionary adaptability of the light sensory function. At the same time, our data provide guidance for the mutational photocycle tuning of LOV domains, with relevance for the growing field of optogenetics.
Assuntos
Proteínas de Bactérias/química , Luz , Oxigênio/química , Rhodobacteraceae/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Cristalografia por Raios X , Cinética , Mutagênese Sítio-Dirigida , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Conformação Proteica , Pseudomonas putida/química , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Rhodobacteraceae/genética , Rhodobacteraceae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Light, oxygen, voltage (LOV) domains are widely distributed in plants, algae, fungi, bacteria, and represent the photo-responsive domains of various blue-light photoreceptor proteins. Their photocycle involves the blue-light triggered adduct formation between the C(4a) atom of a non-covalently bound flavin chromophore and the sulfur atom of a conserved cysteine in the LOV sensor domain. LOV proteins show considerable variation in the structure of N- and C-terminal elements which flank the LOV core domain, as well as in the lifetime of the adduct state. RESULTS: Here, we report the photochemical, structural and functional characterization of DsLOV, a LOV protein from the photoheterotrophic marine α-proteobacterium Dinoroseobacter shibae which exhibits an average adduct state lifetime of 9.6 s at 20°C, and thus represents the fastest reverting bacterial LOV protein reported so far. Mutational analysis in D. shibae revealed a unique role of DsLOV in controlling the induction of photopigment synthesis in the absence of blue-light. The dark state crystal structure of DsLOV determined at 1.5 Å resolution reveals a conserved core domain with an extended N-terminal cap. The dimer interface in the crystal structure forms a unique network of hydrogen bonds involving residues of the N-terminus and the ß-scaffold of the core domain. The structure of photoexcited DsLOV suggests increased flexibility in the N-cap region and a significant shift in the Cα backbone of ß strands in the N- and C-terminal ends of the LOV core domain. CONCLUSIONS: The results presented here cover the characterization of the unusual short LOV protein DsLOV from Dinoroseobacter shibae including its regulatory function, extremely fast dark recovery and an N-terminus mediated dimer interface. Due to its unique photophysical, structural and regulatory properties, DsLOV might thus serve as an alternative model system for studying light perception by LOV proteins and physiological responses in bacteria.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Rhodobacteraceae/química , Organismos Aquáticos/química , Organismos Aquáticos/crescimento & desenvolvimento , Cristalização/métodos , Cristalografia por Raios X , Análise Mutacional de DNA , Modelos Moleculares , Processos Fototróficos , Pigmentos Biológicos/metabolismo , Conformação Proteica , Multimerização Proteica , Rhodobacteraceae/crescimento & desenvolvimentoRESUMO
Light-Oxygen-Voltage (LOV) flavoproteins transduce a light signal into variable signaling outputs via a structural rearrangement in the sensory core domain, which is then relayed to fused effector domains via α-helical linker elements. Short LOV proteins from Pseudomonadaceae consist of a LOV sensory core and N- and C-terminal α-helices of variable length, providing a simple model system to study the molecular mechanism of allosteric activation. Here we report the crystal structures of two LOV proteins from Pseudomonas fluorescens - SBW25-LOV in the fully light-adapted state and Pf5-LOV in the dark-state. In a comparative analysis of the Pseudomonadaceae short LOVs, the structures demonstrate light-induced rotation of the core domains and splaying of the proximal A'α and Jα helices in the N and C-termini, highlighting evidence for a conserved signal transduction mechanism. Another distinguishing feature of the Pseudomonadaceae short LOV protein family is their highly variable dark recovery, ranging from seconds to days. Understanding this variability is crucial for tuning the signaling behavior of LOV-based optogenetic tools. At 37 °C, SBW25-LOV and Pf5-LOV exhibit adduct state lifetimes of 1470 min and 3.6 min, respectively. To investigate this remarkable difference in dark recovery rates, we targeted three residues lining the solvent channel entrance to the chromophore pocket where we introduced mutations by exchanging the non-conserved amino acids from SBW25-LOV into Pf5-LOV and vice versa. Dark recovery kinetics of the resulting mutants, as well as MD simulations and solvent cavity calculations on the crystal structures suggest a correlation between solvent accessibility and adduct lifetime.
Assuntos
Proteínas de Bactérias , Flavoproteínas , Fotorreceptores Microbianos , Pseudomonas fluorescens , Luz , Oxigênio , Transdução de Sinais , Solventes , Flavoproteínas/química , Flavoproteínas/genética , Flavoproteínas/metabolismo , Domínios Proteicos , Conformação Proteica em alfa-Hélice , Pseudomonas fluorescens/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Optogenética , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Mutação , Cristalografia por Raios XRESUMO
The dynamic behavior of Protein Disulfide Isomerase (PDI) in an aqueous solution environment under physiologically active pH has been experimentally verified in this study using Small Angle X-ray Scattering (SAXS) technique. The structural mechanism of dimerization for full-length PDI molecules and co-complex with two renowned substrates has been comprehensively discussed. The structure models obtained from the SAXS data of PDI purified from bovine liver display behavior duality between unaccompanied-enzyme and after engaged with substrates. The analysis of SAXS data revealed that PDI exists as a homo-dimer in the solution environment, and substrate induction provoked its segregation into monomer to enable the enzyme to interact systematically with incoming clients. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00198-0.
RESUMO
Pseudomonas species have become promising cell factories for the production of natural products due to their inherent robustness. Although these bacteria have naturally evolved strategies to cope with different kinds of stress, many biotechnological applications benefit from engineering of optimised chassis strains with specially adapted tolerance traits. Here, we explored the formation of outer membrane vesicles (OMV) of Pseudomonas putida KT2440. We found OMV production to correlate with the recombinant production of a natural compound with versatile beneficial properties, the tripyrrole prodigiosin. Further, several P. putida genes were identified, whose up- or down-regulated expression allowed controlling OMV formation. Finally, genetically triggering vesiculation in production strains of the different alkaloids prodigiosin, violacein, and phenazine-1-carboxylic acid, as well as the carotenoid zeaxanthin, resulted in up to three-fold increased product yields. Consequently, our findings suggest that the construction of robust strains by genetic manipulation of OMV formation might be developed into a useful tool which may contribute to improving limited biotechnological applications.
Assuntos
Produtos Biológicos , Pseudomonas putida , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Prodigiosina/metabolismo , Produtos Biológicos/metabolismo , Biotecnologia , Zeaxantinas/metabolismoRESUMO
Human C-C motif ligand 16 (CCL16) is a chemokine that is distinguished by a large cleavable C-terminal extension of unknown significance. Conflicting data have been reported concerning its tissue distribution and modulation of expression, rendering the biological function of CCL16 enigmatic. Here, we report an integrated approach to the characterisation of this chemokine, including a re-assessment of its expression characteristics as well as a biophysical investigation with respect to its structure and dynamics. Our data indicate that CCL16 is chiefly synthesised by hepatocytes, without an appreciable response to mediators of inflammation, and circulates in the blood as a full-length protein. While the crystal structure of CCL16 confirms the presence of a canonical chemokine domain, molecular dynamics simulations support the view that the C-terminal extension impairs the accessibility of the glycosaminoglycan binding sites and may thus serve as an intrinsic modulator of biological activity.
Assuntos
Quimiocinas CC , Quimiocinas , Humanos , Quimiocinas CC/metabolismo , Ligantes , GlicosaminoglicanosRESUMO
Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa ΔplaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-Å-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the PlaF-mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.
Assuntos
Fosfolipídeos , Pseudomonas aeruginosa , Proteínas de Bactérias/genética , Glicerofosfolipídeos/metabolismo , Proteínas de Membrana , Fosfolipases A , Pseudomonas aeruginosa/metabolismo , Fatores de Virulência/metabolismoRESUMO
The primary photochemistry is similar among the flavin-bound sensory domains of light-oxygen-voltage (LOV) photoreceptors, where upon blue-light illumination a covalent adduct is formed on the microseconds time scale between the flavin chromophore and a strictly conserved cysteine residue. In contrast, the adduct-state decay kinetics vary from seconds to days or longer. The molecular basis for this variation among structurally conserved LOV domains is not fully understood. Here, we selected PpSB2-LOV, a fast-cycling (τrec 3.5 min, 20 °C) short LOV protein from Pseudomonas putida that shares 67% sequence identity with a slow-cycling (τrec 2467 min, 20 °C) homologous protein PpSB1-LOV. Based on the crystal structure of the PpSB2-LOV in the dark state reported here, we used a comparative approach, in which we combined structure and sequence information with molecular dynamic (MD) simulations to address the mechanistic basis for the vastly different adduct-state lifetimes in the two homologous proteins. MD simulations pointed toward dynamically distinct structural region, which were subsequently targeted by site-directed mutagenesis of PpSB2-LOV, where we introduced single- and multisite substitutions exchanging them with the corresponding residues from PpSB1-LOV. Collectively, the data presented identify key amino acids on the Aß-Bß, Eα-Fα loops, and the Fα helix, such as E27 and I66, that play a decisive role in determining the adduct lifetime. Our results additionally suggest a correlation between the solvent accessibility of the chromophore pocket and adduct-state lifetime. The presented results add to our understanding of LOV signaling and will have important implications in tuning the signaling behavior (on/off kinetics) of LOV-based optogenetic tools.
Assuntos
Proteínas de Bactérias/química , Oxigênio/química , Pseudomonas putida/metabolismo , Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Oxigênio/metabolismo , Processos Fotoquímicos , Conformação ProteicaRESUMO
Src homology 3 (SH3) domains are mediators of protein-protein interactions. They comprise approximately 60 amino acid residues and are found in many intracellular signaling proteins. Here, we present the crystal structure of the SH3 domain from phosphatidylinositol 3-kinase (PI3K) in complex with the 12-residue proline-rich peptide PD1R (HSKRPLPPLPSL). The crystal structure of the PI3K SH3-PD1R complex at a resolution of 1.7 A reveals type I ligand orientation of the bound peptide with an extended conformation where the central portion forms a left-handed type II polyproline (PPII) helix. The overall structure of the SH3 domain shows minimal changes on ligand binding. In addition, we also attempted crystallization with another peptide ligand (PD1) where the residue at anchor position P(-3) is a tyrosine. The crystals obtained did not contain the PD1 ligand; instead, the ligand binding site is partially occupied by residues Arg18 and Trp55 from the symmetry-related PI3K SH3 molecule. Considering these crystal structures of PI3K SH3 together with published reports, we provide a comparative analysis of protein-ligand interactions that has helped us identify the individual residues which play an important role in defining target specificity.
Assuntos
Oligopeptídeos/metabolismo , Peptídeos/química , Fosfatidilinositol 3-Quinases/química , Domínios de Homologia de src/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Ligação ProteicaRESUMO
Termination of the G-protein-coupled receptor signaling involves phosphorylation of its C-terminus and subsequent binding of the regulatory protein arrestin. In the visual system, arrestin-1 preferentially binds to photoactivated and phosphorylated rhodopsin and inactivates phototransduction. Here, we have investigated binding of a synthetic phosphopeptide of bovine rhodopsin (residues 323-348) to the active variants of visual arrestin-1: splice variant p44, and the mutant R175E. Unlike the wild type arrestin-1, both these arrestins are monomeric in solution. Solution structure analysis using small angle X-ray scattering supported by size exclusion chromatography results reveal dimerization in both the arrestins in the presence of phosphopeptide. Our results are the first report, to our knowledge, on receptor-induced oligomerization in arrestin, suggesting possible roles for the cellular function of arrestin oligomers. Given high structural homology and the similarities in their activation mechanism, these results are expected to have implications for all arrestin isoforms.
Assuntos
Arrestina/química , Arrestina/metabolismo , Multimerização Proteica , Rodopsina/química , Rodopsina/metabolismo , Animais , Bovinos , Cristalografia por Raios X , Fosforilação , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Human guanylate-binding protein 1 (hGBP1) belongs to the family of dynamin-like proteins and is activated by addition of nucleotides, leading to protein oligomerization and stimulated GTPase activity. In vivo, hGBP1 is post-translationally modified by attachment of a farnesyl group yielding farn-hGBP1. In this study, hydrodynamic differences in farn-hGBP1 and unmodified hGBP1 were investigated using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and analytical size-exclusion chromatography (SEC). In addition, we performed small-angle X-ray scattering (SAXS) experiments coupled with a SEC setup (SEC-SAXS) to investigate structural properties of nonmodified hGBP1 and farn-hGBP1 in solution. SEC-SAXS measurements revealed that farnesylation keeps hGBP1 in its inactive monomeric and crystal-like conformation in nucleotide-free solution, whereas unmodified hGBP1 forms a monomer-dimer equilibrium both in the inactive ground state in nucleotide-free solution as well as in the activated state that is trapped by addition of the nonhydrolysable GTP analogue GppNHp. Nonmodified hGBP1 is structurally perturbed as compared to farn-hGBP. In particular, GppNHp binding leads to large structural rearrangements and higher conformational flexibility of the monomer and the dimer. Structural changes observed in the nonmodified protein are prerequisites for further oligomer assemblies of farn-hGBP1 that occur in the presence of nucleotides. DATABASE: All SEC-SAXS data, corresponding fits to the data and structural models are deposited in the Small Angle Scattering Biological Data Bank [SASBDB (Nucleic Acids Res, 43, 2015, D357)] with project IDs: SASDEE8, SASDEF8, SASDEG8, SASDEH8, SASDEJ8, SASDEK8, SASDEL8 and SASDEM8.
Assuntos
Proteínas de Ligação ao GTP/química , Prenilação , Multimerização Proteica , Cromatografia , Difusão Dinâmica da Luz , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The human membrane-bound α/ß-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane-bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2-AG and esters of medium-length fatty acids. PA2949 in the presence of n-octyl ß-D-glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified from Escherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space group I4122 and diffracted to a resolution of 2.54â Å. This study provides hints on the functional similarity of ABHD6-like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult-to-crystallize proteins.
Assuntos
Esterases/química , Esterases/isolamento & purificação , Monoacilglicerol Lipases/química , Pseudomonas aeruginosa/enzimologia , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Cristalização , Estabilidade Enzimática , Humanos , Cinética , Especificidade por Substrato , TemperaturaRESUMO
Sterile alpha motif (SAM) domains are protein interaction modules that are involved in a diverse range of biological functions such as transcriptional and translational regulation, cellular signalling, and regulation of developmental processes. SH3 domain-containing protein expressed in lymphocytes 1 (SLy1) is involved in immune regulation and contains a SAM domain of unknown function. In this report, the structure of the SLy1 SAM domain was solved and revealed that this SAM domain forms a symmetric homodimer through a novel interface. The interface consists primarily of the two long C-terminal helices, α5 and α5', of the domains packing against each other. The dimerization is characterized by a dissociation constant in the lower micromolar range. A SLy1 SAM domain construct with an extended N-terminus containing five additional amino acids of the SLy1 sequence further increases the stability of the homodimer, making the SLy1 SAM dimer two orders of magnitude more stable than previously studied SAM homodimers, suggesting that the SLy1 SAM dimerization is of functional significance. The SLy1 SAM homodimer contains an exposed mid-loop surface on each monomer, which may provide a scaffold for mediating interactions with other SAM domain-containing proteins via a typical mid-loop-end-helix interface.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Multimerização Proteica , Motivo Estéril alfa , Conformação ProteicaRESUMO
Light, oxygen, voltage (LOV) photoreceptors consist of conserved photo-responsive domains in bacteria, archaea, plants and fungi, and detect blue-light via a flavin cofactor. We investigated the blue-light induced conformational transition of the dimeric photoreceptor PpSB1-LOV-R66I from Pseudomonas putida in solution by using small-angle X-ray scattering (SAXS). SAXS experiments of the fully populated light- and dark-states under steady-state conditions revealed significant structural differences between the two states that are in agreement with the known structures determined by crystallography. We followed the transition from the light- to the dark-state by using SAXS measurements in real-time. A two-state model based on the light- and dark-state conformations could describe the measured time-course SAXS data with a relaxation time τREC of ~ 34 to 35 min being larger than the recovery time found with UV/vis spectroscopy. Unlike the flavin chromophore-based UV/vis method that is sensitive to the local chromophore environment in flavoproteins, SAXS-based assay depends on protein conformational changes and provides with an alternative to measure the recovery kinetics.
Assuntos
Flavoproteínas/metabolismo , Oxigênio/metabolismo , Fotorreceptores Microbianos/metabolismo , Pseudomonas putida/metabolismo , Espalhamento a Baixo Ângulo , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Mononucleotídeo de Flavina/química , Cinética , Domínios Proteicos , Estrutura Secundária de Proteína , Espectrofotometria Ultravioleta , Difração de Raios XRESUMO
Protein purity and yield are two critical parameters for successful protein characterization using structural techniques such as X-ray crystallography, NMR, and several other biophysical methods. The yeast Saccharomyces cerevisiae is one of the popular eukaryotic model systems for overexpression and subsequent purification of recombinant proteins. Here, we describe a protocol for cloning, overexpression, purification, and crystallization of arrestin-1 and its splice variant p44 from yeast. The purification protocol involves a single-affinity chromatography step on a Strep-Tactin column. Highly purified arrestins can be concentrated up to 15mg/mL using ultrafiltration and can be stored in the frozen state for several months without any loss of functionality.
Assuntos
Arrestina/química , Arrestina/isolamento & purificação , Cromatografia de Afinidade/métodos , Saccharomyces cerevisiae/metabolismo , Arrestina/genética , Cromatografia de Afinidade/instrumentação , Cristalização/métodos , Processamento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , UltrafiltraçãoRESUMO
Unique features of Light-Oxygen-Voltage (LOV) proteins like relatively small size (~12-19 kDa), inherent modularity, highly-tunable photocycle and oxygen-independent fluorescence have lately been exploited for the generation of optical tools. Structures of LOV domains reported so far contain a flavin chromophore per protein molecule. Here we report two new findings on the short LOV protein W619_1-LOV from Pseudomonas putida. First, the apo-state crystal structure of W619_1-LOV at 2.5 Å resolution reveals conformational rearrangements in the secondary structure elements lining the chromophore pocket including elongation of the Fα helix, shortening of the Eα-Fα loop and partial unfolding of the Eα helix. Second, the apo W619_1-LOV protein binds both natural and structurally modified flavin chromophores. Remarkably different photophysical and photochemical properties of W619_1-LOV bound to 7-methyl-8-chloro-riboflavin (8-Cl-RF) and lumichrome imply application of these variants as novel optical tools as they offer advantages such as no adduct state formation, and a broader choice of wavelengths for in vitro studies.
Assuntos
Proteínas de Bactérias/química , Pseudomonas putida/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de FluorescênciaRESUMO
Pseudomonas aeruginosa strain 1001 produces an esterase (EstA) that can hydrolyse the racemic methyl ester of ß-acetylthioisobutyrate to produce the (D)-enantiomer, which serves as a precursor of captopril, a drug used for treatment of hypertension. We show here that PA2949 from P. aeruginosa PA01, a homologue of EstA, can efficiently be expressed in an enzymatically active form in E. coli. The enzyme is membrane-associated as demonstrated by cell fractionation studies. PA2949 was purified to homogeneity after solubilisation with the nonionic detergent, Triton X-100, and was shown to possess a conserved esterase catalytic triad consisting of Ser137-His258-Asp286. Our results should allow the development of an expression and purification strategy to produce this biotechnologically relevant esterase in a pure form with a high yield.
RESUMO
Light-Oxygen-Voltage (LOV) domains represent the photo-responsive domains of various blue-light photoreceptor proteins and are widely distributed in plants, algae, fungi, and bacteria. Here, we report the dark-state crystal structure of PpSB1-LOV, a slow-reverting short LOV protein from Pseudomonas putida that is remarkably different from our previously published "fully light-adapted" structure [1]. A direct comparison of the two structures provides insight into the light-activated signaling mechanism. Major structural differences involve a~11Å movement of the C terminus in helix Jα, ~4Å movement of Hß-Iß loop, disruption of hydrogen bonds in the dimer interface, and a~29° rotation of chain-B relative to chain-A as compared to the light-state dimer. Both crystal structures and solution NMR data are suggestive of the key roles of a conserved glutamine Q116 and the N-cap region consisting of A'α-Aß loop and the A'α helix in controlling the light-activated conformational changes. The activation mechanism proposed here for the PpSB1-LOV supports a rotary switch mechanism and provides insights into the signal propagation mechanism in naturally existing and artificial LOV-based, two-component systems and regulators.