Lipases comprise a large class of hydrolytic enzymes which catalyze the cleavage of the ester bonds in triacylglycerols and find numerous biotechnological applications. Previously, we have cloned the ...gene coding for a novel esterase PMGL2 from a Siberian permafrost metagenomic DNA library. We have determined the 3D structure of PMGL2 which belongs to the hormone-sensitive lipase (HSL) family and contains a new variant of the active site motif, GCSAG. Similar to many other HSLs, PMGL2 forms dimers in solution and in the crystal. Our results demonstrated that PMGL2 and structurally characterized members of the GTSAG motif subfamily possess a common dimerization interface that significantly differs from that of members of the GDSAG subfamily of known structure. Moreover, PMGL2 had a unique organization of the active site cavity with significantly different topology compared to the other lipolytic enzymes from the HSL family with known structure including the distinct orientation of the active site entrances within the dimer and about four times larger size of the active site cavity. To study the role of the cysteine residue in GCSAG motif of PMGL2, the catalytic properties and structure of its double C173T/C202S mutant were examined and found to be very similar to the wild type protein. The presence of the bound PEG molecule in the active site of the mutant form allowed for precise mapping of the amino acid residues forming the substrate cavity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
This Special Issue of Biomolecules demonstrates the almost unlimited possibilities of modern protein engineering in gene expression, protein production and modification, as well as the design and ...creation of new proteins ...
Microbial rhodopsins comprise a diverse family of retinal-containing membrane proteins that convert absorbed light energy to transmembrane ion transport or sensory signals. Incorporation of these ...proteins in proteoliposomes allows their properties to be studied in a native-like environment; however, unidirectional protein orientation in the artificial membranes is rarely observed. We aimed to obtain proteoliposomes with unidirectional orientation using a proton-pumping retinal protein from
, ESR, as a model. Three ESR hybrids with soluble protein domains (mCherry or thioredoxin at the C-terminus and Caf1M chaperone at the N-terminus) were obtained and characterized. The photocycle of the hybrid proteins incorporated in proteoliposomes demonstrated a higher pK
of the M state accumulation compared to that of the wild-type ESR. Large negative electrogenic phases and an increase in the relative amplitude of kinetic components in the microsecond time range in the kinetics of membrane potential generation of ESR-Cherry and ESR-Trx indicate a decrease in the efficiency of transmembrane proton transport. On the contrary, Caf-ESR demonstrates a native-like kinetics of membrane potential generation and the corresponding electrogenic stages. Our experiments show that the hybrid with Caf1M promotes the unidirectional orientation of ESR in proteoliposomes.
ESR, a light-driven proton pump from Exiguobacterium sibiricum, contains a lysine residue (Lys96) in the proton donor site. Substitution of Lys96 with a nonionizable residue greatly slows ...reprotonation of the retinal Schiff base. The recent study of electrogenicity of the K96A mutant revealed that overall efficiency of proton transport is decreased in the mutant due to back reactions (Siletsky et al., BBA, 2019). Similar to members of the proteorhodopsin and xanthorhodopsin families, in ESR the primary proton acceptor from the Schiff base, Asp85, closely interacts with His57. To examine the role of His57 in the efficiency of proton translocation by ESR, we studied the effects of H57N and H57N/K96A mutations on the pH dependence of light-induced pH changes in suspensions of Escherichia coli cells, kinetics of absorption changes and electrogenic proton transfer reactions during the photocycle. We found that at low pH (<5) the proton pumping efficiency of the H57N mutant in E. coli cells and its electrogenic efficiency in proteoliposomes is substantially higher than in the WT, suggesting that interaction of His57 with Asp85 sets the low pH limit for H+ pumping in ESR. The electrogenic components that correspond to proton uptake were strongly accelerated at low pH in the mutant indicating that Lys96 functions as a very efficient proton donor at low pH. In the H57N/K96A mutant, a higher H+ pumping efficiency compared with K96A was observed especially at high pH, apparently from eliminating back reactions between Asp85 and the Schiff base by the H57N mutation.
•ESR is a proton pump with Lys96 at the site of the H+ donor to the Schiff base (SB).•His57 near H+ acceptor (Asp85) controls proton affinity of the latter.•At low pH, the H57N mutant pumps protons more efficiently than the WT.•H57N mutation eliminates/decreases back reactions between Asp85 and the SB.•Lys96 acts as a fast and efficient donor at neutral and low pH.
Cell-surface display using anchor motifs of outer membrane proteins allows exposure of target peptides and proteins on the surface of microbial cells. Previously, we obtained and characterized highly ...catalytically active recombinant oligo-α-1,6-glycosidase from the psychrotrophic bacterium
Exiguobacterium sibiricum
(EsOgl). It was also shown that the autotransporter AT877 from
Psychrobacter cryohalolentis
and its deletion variants efficiently displayed type III fibronectin (
10
Fn3) domain 10 on the surface of
Escherichia coli
cells. The aim of the work was to obtain an AT877-based system for displaying EsOgl on the surface of bacterial cells. The genes for the hybrid autotransporter EsOgl877 and its deletion mutants EsOgl877Δ239 and EsOgl877Δ310 were constructed, and the enzymatic activity of EsOgl877 was investigated. Cells expressing this protein retained ~90% of the enzyme maximum activity within a temperature range of 15-35°C. The activity of cells expressing EsOgl877Δ239 and EsOgl877Δ310 was 2.7 and 2.4 times higher, respectively, than of the cells expressing the full-size AT. Treatment of cells expressing EsOgl877 deletion variants with proteinase K showed that the passenger domain localized to the cell surface. These results can be used for further optimization of display systems expressing oligo-α-1,6-glycosidase and other heterologous proteins on the surface of
E. coli
cells.
The primary stages of the Exiguobacterium sibiricum rhodopsin (ESR) photocycle were investigated by femtosecond absorption laser spectroscopy in the spectral range of 400–900 nm with a time ...resolution of 25 fs. The dynamics of the ESR photoreaction were compared with the reactions of bacteriorhodopsin (bR) in purple membranes (bRPM) and in recombinant form (bRrec). The primary intermediates of the ESR photocycle were similar to intermediates I , J , and K in bacteriorhodopsin photoconversion. The CONTIN program was applied to analyze the characteristic times of the observed processes and to clarify the reaction scheme. A similar photoreaction pattern was observed for all studied retinal proteins, including two consecutive dynamic Stokes shift phases lasting ∼0.05 and ∼0.15 ps. The excited state decays through a femtosecond reactive pathway, leading to retinal isomerization and formation of product J , and a picosecond nonreactive pathway that leads only to the initial state. Retinal photoisomerization in ESR takes 0.69 ps, compared with 0.48 ps in bRPM and 0.74 ps in bRrec. The nonreactive excited state decay takes 5 ps in ESR and ∼3 ps in bR. We discuss the similarity of the primary reactions of ESR and other retinal proteins.
The progress in optogenetics largely depends on the development of light-activated proteins as new molecular tools. Using cultured hippocampal neurons, we compared the properties of two ...light-activated cation channels – classical channelrhodopsin-2 from
Chlamydomonas reinhardtii
(
Cr
ChR2) and recently described channelrhodopsin isolated from the alga
Platymonas subcordiformis
(
Ps
ChR2).
Ps
ChR2 ensured generation of action potentials by neurons when activated by the pulsed light stimulation with the frequencies up to 40-50 Hz, while the upper limit for
Cr
ChR2 was 20-30 Hz. An important advantage of
Ps
ChR2 compared to classical channelrhodopsin
Cr
ChR2 is the blue shift of its excitation spectrum, which opens the possibility for its application in all-optical electrophysiology experiments that require the separation of the maxima of the spectra of channelrhodopsins used for the stimulation of neurons and the maxima of the excitation spectra of various red fluorescent probes. We compared the response (generation of action potentials) of neurons expressing
Cr
ChR2 and
Ps
ChR2 to light stimuli at 530 and 550 nm commonly used for the excitation of red fluorescent probes. The 530-nm light was significantly (3.7 times) less efficient in the activation of neurons expressing
Ps
ChR2 vs.
Cr
ChR2-expressing neurons. The light at 550 nm, even at the maximal used intensity, failed to stimulate neurons expressing either of the studied opsins. This indicates that the
Ps
ChR2 channelrhodopsin from the alga
P. subcordiformis
is a promising optogenetic tool, both in terms of its frequency characteristics and possibility of its application for neuronal stimulation with a short-wavelength (blue, 470 nm) light accompanied by simultaneous recording of various physiological processes using fluorescent probes.
Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in ...cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K+ channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid–protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.
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► Protein/nanodisc complexes can be assembled using SDS denatured membrane proteins. ► Obtained complexes enclose the folded polytopic and multimeric membrane domains. ► Nanodiscs promote in vitro folding of the 7-helical bacteriorhodopsin ESR. ► Nanodiscs promote in vitro folding of the homotetrameric K+ channel KcsA. ► Optimization of lipid composition is required to achieve efficient folding
Retinal-containing light-sensitive proteins – rhodopsins – are found in many microorganisms. Interest in them is largely explained by their role in light energy storage and photoregulation in ...microorganisms, as well as the prospects for their use in optogenetics to control neuronal activity, including treatment of various diseases. One of the representatives of microbial rhodopsins is ESR, the retinal protein of
Exiguobacterium sibiricum
. What distinguishes ESR from homologous proteins is the presence of a lysine residue (Lys96) as a proton donor for the Schiff base. This feature, along with the hydrogen bond of the proton acceptor Asp85 with the His57 residue, determines functional characteristics of ESR as a proton pump. This review examines the results of ESR studies conducted using various methods, including direct electrometry. Comparison of the obtained data with the results of structural studies and with other retinal proteins allows us to draw conclusions about the mechanisms of transport of hydrogen ions in ESR and similar retinal proteins.
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