D. A. Dolgikh

Breaking the Carboxyl Rule LYSINE 96 FACILITATES REPROTONATION OF THE SCHIFF BASE IN THE PHOTOCYCLE OF A RETINAL PROTEIN FROM EXIGUOBACTERIUM SIBIRICUM

Background: Lysine rather than a carboxylic residue is in place of the internal proton donor in the E. sibiricum proton pump. Results: H+ uptake precedes reprotonation of the retinal Schiff base. K96A mutation slows it by >100-fold. Conclusion: Lysine 96 facilitates proton delivery to the Schiff base. Significance: This is the first example where lysine mediates proton transfer to the retinal Schiff base. A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ϵ-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.

Photocycle of Exiguobacterium sibiricum rhodopsin characterized by low-temperature trapping in the IR and time-resolved studies in the visible.

The photocycle of the retinal protein from Exiguobacterium sibiricum, which differs from bacteriorhodopsin in both its primary donor and acceptor, is characterized by visible and infrared spectroscopy. At pH above pKa ~6.5, we find a bacteriorhodopsin-like photocycle, which originates from excitation of the all-trans retinal chromophore with K-, L-, M-, and N-like intermediates. At pH below pKa ~6.5, the M state, which reflects Schiff base deprotonation during proton pumping, is not accumulated. However, using the infrared band at ~1760 cm(-1) as a marker for transient protonation of the primary acceptor, we find that Schiff base deprotonation must have occurred at pH not only above but also below the pKa ~6.5. Thus, the M state is formed but not accumulated for kinetic reasons. Further, chromophore reisomerization from the 13-cis to the all-trans conformation occurs very late in the photocycle. The strongly red-shifted states that dominate the second half of the cycle are produced before the reisomerization step, and by this criterion, they are not O-like but rather N-like states. The assignment of photocycle intermediates enables reevaluation of the photocycle; its specific features are discussed in relation to the general mechanism of proton transport in retinal proteins.

Characterization of a cold-active lipase from Psychrobacter cryohalolentis K5T and its deletion mutants

A gene coding for cold-active lipase from the psychrotrophic Gram-negative bacterium Psychrobacter cryohalolentis K5T isolated from a Siberian cryopeg has been cloned and expressed in Escherichia coli. The recombinant protein Lip1Pc with a 6× histidine tag at its C-terminus was purified by nickel affinity chromatography. With p-nitrophenyl dodecanoate (C12) as a substrate, the purified recombinant protein displayed maximum lipolytic activity at 25°C and pH 8.0. Increasing the temperature above 40°C and addition of various metal ions and organic solvents inhibited the enzymatic activity of Lip1Pc. Most nonionic detergents, such as Triton X-100 and Tween 20, slightly increased the lipase activity, while SDS completely inhibited it. To investigate the functional significance of the Lip1Pc N-terminal domain, we constructed five deletion mutants of this protein. The ND1 and ND2 mutants displayed specific activity reduced by 30–35%, while other truncated proteins were completely inactive. Both mutants demonstrated increased activity towards p-nitrophenyl decanoate (C10) and impaired utilization of C16 substrate. Although optimum reaction temperature of ND2 lowered to 20°C, it displayed enhanced stability by 44% after incubation at 40°C. The results prove that the N-terminal domain of Lip1Pc has a fundamental impact on the activity and stability of the protein.

Bacterial expression of the water-soluble domain of lynx1, an endogenous neuromodulator of human nicotinic receptors

The membrane-associated protein Lynx1 is expressed in the human central nervous system and plays an important role in the regulation of the activity of nicotinic acetylcholine receptors. In the present study, ws-Lynx1 encoding the water-soluble domain of human Lynx1 was cloned and studied by heterological expression in E. coli. In milligram quantities, the ws-Lynx1 protein could only be obtained in cytoplasmic inclusion bodies of bacterial cells. To optimize the yield of ws-Lynx1 recombinant protein, a system was developed that allowed retrieving functionally active ws-Lynx1 from the inclusion bodies. After renaturation, the protein was characterized by mass spectrometry and by circular dichroism spectroscopy. ws-Lynx1 was shown to inhibit the binding of [125I]-α-bungarotoxin to membranes from the electric organ of the Torpedo californica ray containing muscle-type nicotinic acetylcholine receptors (α12βγδ) in a competitive manner.

In vitro production of three-finger neurotoxins from snake venoms, a disulfide rich proteins. Problems and their solutions (Review)

Abstractα-Neurotoxins from snake venom are highly efficient inhibitors of nicotinic acetylcholine receptors (nAChRs). These small proteins with a β-structural organization attract much interest as a tool for studies of nAChR and as prototypes for the development of new pharmaceuticals for the treatment of diseases of the nervous system. However, in vitro production of “three-finger” neurotoxins is complicated by the presence of four or five spatially closed disulfide bonds in their molecules. The present review contains a description of the most frequently used modern approaches for the E. coli expression of recombinant proteins (direct expression, expression as hybrid proteins, and secretion) with an emphasis placed on the recombinant production of snake α-neurotoxins. The methods of E. coli expression of isotopically labeled neurotoxins are described. The proposed solutions will be of broad interest for the bacterial production of other disulfide rich proteins.

Development and characterization of antibodies against aflatoxins

A panel of ten monoclonal antibodies against aflatoxins B1, B2, and G2 was produced and comprehensively characterized. The affinity and cross reactivity of these antibodies were determined using the methods of direct, indirect, and competitive ELISA. The structures of monoclonal antibody genes were comprehensively studied and the variable and constant regions of the antibody genes were cloned and sequenced. Sequencing analysis confirmed the results of isotyping the light and heavy antibody chains obtained by ELISA. Variable and constant fragments of the antibody genes were cloned into a bicistron expression vector for the recombinant Fab-fragment for one of the antibodies expressed in Escherichia coli and purified. Thus, data were obtained that can be useful for the development of an aflatoxin detection system on the basis of the described monoclonal antibodies and the creation of recombinant antibodies with changed parameters of specificity using protein engineering methods.

Electrogenic steps of light-driven proton transport in ESR, a retinal protein from Exiguobacterium sibiricum.

A retinal protein from Exiguobacterium sibiricum (ESR) functions as a light-driven proton pump. Unlike other proton pumps, it contains Lys96 instead of a usual carboxylic residue in the internal proton donor site. Nevertheless, the reprotonation of the Schiff base occurs fast, indicating that Lys96 facilitates proton transfer from the bulk. In this study we examined kinetics of light-induced transmembrane electrical potential difference, ΔΨ, generated in proteoliposomes reconstituted with ESR. We show that total magnitude of ΔΨ is comparable to that produced by bacteriorhodopsin but its kinetic components and their pH dependence are substantially different. The results are in agreement with the earlier finding that proton uptake precedes reprotonation of the Schiff base in ESR, suggesting that Lys96 is unprotonated in the initial state and gains a proton transiently in the photocycle. The electrogenic phases and the photocycle transitions related to proton transfer from the bulk to the Schiff base are pH dependent. At neutral pH, they occur with τ 0.5ms and 4.5ms. At alkaline pH, the fast component ceases and Schiff base reprotonation slows. At pH8.4, a spectrally silent electrogenic component with τ 0.25ms is detected, which can be attributed to proton transfer from the bulk to Lys96. At pH5.1, the amplitude of ΔΨ decreases 10 fold, reflecting a decreased yield and rate of proton transfer, apparently from protonation of the acceptor (Asp85-His57 pair) in the initial state. The features of the photoelectric potential generation correlate with the ESR structure and proposed mechanism of proton transfer.

Human secreted proteins SLURP‐1 and SLURP‐2 control the growth of epithelial cancer cells via interactions with nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are a promising target for development of new anticancer therapies. Here we have investigated the effects of the endogenous human proteins SLURP‐1 and SLURP‐2, antagonists of nAChRs, on human epithelial cancer cells.

Recombinant production and structural studies of the human Lypd6 and Lypd6b proteins

Lypd6 and Lypd6b are human three-finger proteins expressed in various tissues and sharing a high degree of sequence homology (~54%). Unlike other members of the Ly6/uPAR family, both proteins additionally bear extended N- and С-terminal sequences flanking the LU-domain. The role of these sequences is unclear. It is known that Lypd6 can increase the amplitude of nicotine-induced calcium currents in mouse trigeminal ganglion neurons. The Danio rerio fish Lypd6 is involved in the regulation of the Wnt/β-cathenin signaling pathway and the knockdown of the lypd6 expression leads to the impairment of embryonic development. The Lypd6b expression in X. laevis oocytes increased the sensitivity of nicotinic acetylcholine receptors to acetylcholine and increased their desensitization rate. Molecular mechanisms of Lypd6 and Lypd6b action as well as their spatial structures remain unknown. In this work, the genes encoding water-soluble variants of human Lypd6 and Lypd6b lacking N-terminal sequences (rLypd6 and rLypd6b) and Lypd6 bearing the N-terminal sequence (N-rLypd6) were obtained and expressed. The proteins were expressed in cytoplasmic inclusion bodies followed by solubilization under denaturing conditions and renaturation. The renaturation conditions were screened to optimize recombinant protein yields. The analysis of NMR spectra of recombinant N-rLypd6, rLypd6, and rLypd6b demonstrated that N-rLypd6 may not have a structured form. The production of milligram quantities of isotope-labeled rLypd6 and rLypd6b analogues allowed characterization of the secondary structure of these proteins and study of their intramolecular dynamics. It was found that rLypd6 and rLypd6b have structural elements typical for the Ly6/uPAR family, although with some unique features, particularly, an additional disulfide bond in the third loop and helical regions in the first and third loops.

Neutralizing monoclonal and chimeric antibodies to human interferon-γ

Autoimmune diseases are chronic diseases characterized by abnormal immune response directed to the body’s own antigens, which leads to tissue damage and violation of their normal functioning. These diseases often lead to disability and even death of patients. Nowadays, several monoclonal antibodies to proinflammatory cytokines and their receptors are used successfully for targeted therapy of autoimmune diseases. One of the most promising targets for the treatment of autoimmune diseases is the interferon-γ (IFN-γ), a key cytokine in Thl-cell differentiation, macrophage activation and inflammation. Five monoclonal antibodies (mAbs) to human IFN-γ were produced in this work. Affinity of the antibodies to IFN-γ and their ability to suppress the activity induced by IFN-γ were studied in order to develop of a potential therapeutic agent. The mAb F1 was selected for chimerization based on the data of this study. The complex of mAb F1 with IFN-γ had a dissociation constant of 1.7 nM; in addition, the antibody showed a low value of IC90 (8.9 ± 2.0 nM) when suppressing IFN-γ-induced HLA-DR antigen expression on the surface of the cell line U937. Bicistronic vector was designed for production of Fab-fragment of a chimeric antibody F1 chim in E. coli cells. The resulting recombinant chimeric Fab-fragment of F1 antibody neutralized IFN-γ activity in vitro and had a dissociation constant of 1.8 nM.