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RF3 Brassica napus L. (InVigorTM Canola) has been in commercial cultivation since 1997. RF3 B. napus was produced by Agrobacterium-mediated transformation to provide glufosniate ammonium herbicide tolerance to the crop. Canola quality (CQ) varieties of Brassica juncea L. (Czern & Coss), a close relative of B. napus, have been available in Canada for more than 20 years. Currently, BASF Agricultural Solutions is developing CQ RF3 B. juncea via conventional breeding with RF3 B. napus to provide similar herbicide tolerance in the B. juncea species. The insertion event RF3 contains the bar gene (origin Streptomyces hygroscopicus) coding for phosphinothricin acetyl transferase (PAT/bar) protein which confers tolerance to glufosinate-ammonium herbicides. RF3 also contains the barstar gene (origin Bacillus amyloliquefaciens), coding for the Barstar protein, which is an inhibitor of the Barnase protein. In the absence of Barnase, there is no impact of Barstar. Many safety studies have been conducted during the development of the canola quality lines of RF3 B. napus and RF3 B. juncea. This report seeks to compare the two species based on the scientific data generated during the safety assessments of RF3 B. napus and RF3 B. juncea and discusses the utility of using studies with one species to support the safety of the other species both containing the same insertion event.

The C40A/C82A double mutant of barstar has been shown to undergo cold denaturation above the water freezing point. By rapidly applying radio‐frequency power to lossy aqueous samples, refolding of barstar from its cold‐denatured state can be followed by real‐time NMR spectroscopy. Since temperature‐induced unfolding and refolding is reversible for this double mutant, multiple cycling can be utilized to obtain 2D real‐time NMR data. Barstar contains two proline residues that adopt a mix of cis and trans conformations in the low‐temperature‐unfolded state, which can potentially induce multiple folding pathways. The high time resolution real‐time 2D‐NMR measurements reported here show evidence for multiple folding pathways related to proline isomerization, and stable intermediates are populated. By application of advanced heating cycles and state‐correlated spectroscopy, an alternative folding pathway circumventing the rate‐limiting cis‐trans isomerization could be observed. The kinetic data revealed intermediates on both, the slow and the fast folding pathway., A state‐of‐the‐art temperature‐jump probe head is used in combination with real‐time 2D NMR experiments to study the kinetics of folding of a cold‐denatured protein. Barstar contains two prolines that adopt a mix of cis and trans conformations in the cold‐denatured state. The high time resolution measurements reported here show evidence for multiple folding pathways related to proline isomerization.

In preparation for a legal implementation of EU-regulation 1829/2003, the Norwegian Environment Agency (former Norwegian Directorate for Nature Management) has requested the Norwegian Food Safety Authority (NFSA) to give final opinions on all genetically modified organisms (GMOs) and products containing or consisting of GMOs that are authorized in the European Union under Directive 2001/18/EC or Regulation 1829/2003/EC within the Authority’s sectoral responsibility. The Norwegian Food Safety Authority has therefore, by letter dated 13 February 2013 (ref. 2012/150202), requested the Norwegian Scientific Committee for Food Safety (VKM) to carry out scientific risk assessments of 39 GMOs and products containing or consisting of GMOs that are authorized in the European Union. The request covers scope(s) relevant to the Gene Technology Act. The request does not cover GMOs that VKM already has conducted its final risk assessments on. However, the Agency requests VKM to consider whether updates or other changes to earlier submitted assessments are necessary. The genetically modified, glufosinate-tolerant oilseed rape lines MS8, RF3 and MS8 x RF3 (Notification C/BE/96/01) are approved under Directive 2001/18/EC for import and processing, for feed and industrial purposes since 26 March 2007 (Commission Decision 2007/232/EC). In addition, processed oil from genetically modified oilseed rape derived from MS8, RF3 and MS8 x RF3 were notified as existing food according to Art. 5 of Regulation (EC) No 258/97 on novel foods and novel food ingredients in November 1999. Existing feed and feed products containing, consisting of or produced from MS8, RF3 and MS8 x RF3 were notified according to Articles 8 and 20 of Regulation (EC) No 1829/2003 and were placed on the market in January 2000. An application for renewal of the authorisation for continued marketing of existing food, food ingredients and feed materials produced from MS8, RF3 and MS8 x RF3 was submitted within the framework of Regulation (EC) No 1829/2003 in June 2007 (EFSA/GMO/RX/MS8/RF3). In addition, an application covering food containing or consisting of, and food produced from or containing ingredients produced from oilseed rape MS8, RF3 and MS8 x RF3 (with the exception of processed oil) was delivered by Bayer CropScience in June 2010 (EFSA/GMO/BE/2010/81). The VKM GMO Panel has previously issued a scientific opinion related to the notification C/BE/96/01 for the placing on the market of the oilseed rape lines for import, processing and feed uses (VKM 2008). The food/feed and environmental risk assessment was commissioned by the Norwegian Environment Agency in connection with the national finalisation of the procedure of the notification C/BE/96/01 in 2008. Due to the publication of updated guidelines for risk assessments of genetically modified plants and new scientific literature, the VKM GMO Panel has decided to deliver an updated food, feed and environmental risk assessment of oilseed rape MS8, RF3 and MS8 x RF3. A scientific opinion on an application for the placing on the market of MS8 x RF3 for food containing or consisting of, and food produced from or containing ingredients produced from MS8 x RF3 (with the exception of processed oil) (EFSA/GMO/BE/2010/81) have also been submitted by the VKM GMO Panel (VKM 2012, unpublished). The risk assessment of the oilseed rape MS8, RF3 and MS8 x RF3 is based on information provided by the notifier in the applications EFSA/GMO/RX/MS8/RF3, EFSA/GMO/BE/2010/81, the notification C/BE/96/01, and scientific comments from EFSA and other member states made available on the EFSA website GMO Extranet. The risk assessment also considered other peer-reviewed scientific literature as relevant. The VKM GMO Panel has evaluated MS8, RF3 and MS8 x RF3 with reference to its intended uses in the European Economic Area (EEA), and according to the principles described in the Norwegian Gene Technology Act and regulations relating to impact assessment pursuant to the Gene Technology Act, Directive 2001/18/EC on the deliberate release into the environment of genetically modified organisms, and Regulation (EC) No 1829/2003 on genetically modified food and feed. The Norwegian Scientific Committee for Food Safety has also decided to take account of the appropriate principles described in the EFSA guidelines for the risk assessment of GM plants and derived food and feed (EFSA 2006, 2011a), the environmental risk assessment of GM plants (EFSA 2010a) and the selection of comparators for the risk assessment of GM plants (EFSA 2011b). The scientific risk assessment of oilseed rape MS8, RF3 and MS8 x RF3 include molecular characterisation of the inserted DNA and expression of target proteins, comparative compositional assessment, food/feed safety assessment, comparative assessment of agronomic and phenotypic characteristics, unintended effects on plant fitness and potential for horizontal and vertical gene transfer. In line with its mandate, VKM emphasised that assessments of sustainable development, societal utility and ethical considerations, according to the Norwegian Gene Technology Act and Regulations relating to impact assessment pursuant to the Gene Technology Act, shall not be carried out by the Panel on Genetically Modified Organisms. The genetically modified oilseed rape lines MS8 and RF3 were developed to provide a pollination control system for production of F1-hybrid seeds (MS8 x RF3). Oilseed rape is a crop capable of undergoing both self-pollination (70%) as well as cross-pollination (30%). Therefore a system to ensure only cross-pollination is required for producing hybrids from two distinct parents. As a result of hybrid vigor cross-pollinated plants produce higher yield as compared to self-pollinating rape. The hybrid system is achieved using a pollination control system by insertion and expression of barnase and barstar genes derived from the soil bacterium Bacillus amyloliquefaciens into two separate transgenic oilseed rape lines. The barnase gene in the male sterile line MS8 encode a ribonuclease peptide (RNase), expressed in the tapetum cells during anther development. The RNase effect RNA levels, disrupting normal cell function, arresting early anther development, and results in the lack of viable pollen and male sterility. The fertility restoration line RF3 contains a barstar gene, coding for a ribonuclease inhibitor (Barstar peptide) expressed only in the tapetum cells of the pollen during anther development. The peptide specifically inhibits the Barnase RNase expressed by the MS8 line. The RNase and the ribonuclease inhibitor form a stable one-to-one complex, in which the RNase is inactivated. As a result, when pollen from the receptor line RF3 is crossed to the male sterile line MS8, the MS8 x RF3 progeny expresses the RNase inhibitor in the tapetum cells of the anthers allowing hybrid plants to develop normal anthers and restore fertility. The barnase and barstar genes in MS8 and RF3 are each linked with the bar gene from Streptomyces hygroscopus. The bar gene is driven by a plant promoter that is active in all green tissues of the plant, and encodes the enzyme phosphinothricin acetyltransferase (PAT). The PAT enzyme inactivates phosphinothricin (PPT), the active constituent of the non-selective herbicide glufosinate-ammonium. The bar gen were transferred to the oilseed rape plants as markers both for use during in vitro selection and as a breeding selection tool in seed production. Molecular Characterisation: The oilseed rape hybrid MS8 x RF3 is produced by conventional crossing. The parental lines MS8 and RF3 are well described in the documentation provided by the applicant, and a number of publications support their data. It seems likely that MS8 contains a complete copy of the desired T-DNA construct including the bar and barnase genes. Likewise, the event RF3 is likely to contain complete copies of the bar and barstar genes in addition to a second incomplete non-functional copy of the bar-gene. The inserts in the single events are preserved in the hybrid MS8 x RF3, and the desired traits are stably inherited over generations. The GMO Panel finds the characterisation of the physical, chemical and functional properties of the recombinant inserts in the oilseed rape transformation events MS8, RF3 and MS8 x RF3 to be satisfactory. The GMO Panel has not identified any novel risks associated with the modified plants based on the molecular characterisation of the inserts. Comparative Assessment: Based on results from comparative analyses of data from field trials located at representative sites and environments in Europe and Canada, it is concluded that oilseed rape MS8, RF3 and MS8 x RF3 is compositionally, agronomically and phenotypically equivalent to the conventional counterpart, except for the newly expressed barnase, barstar and PAT proteins. In the Canadian field trials, however, compositional and phenotypic characteristics of oilseed rape MS8, RF3 and MS8 x RF3 were compared to null-segregant comparators. As negative segregants are derived from a GM organism, the VKM GMO Panel does not consider them appropriate conventional counterparts with a history of safe use. Data obtained from field trials with negative segregants were considered as supplementary information for the RA. Based on the assessment of available data, the VKM GMO Panel is of the opinion that conventional crossing of oilseed rape MS8 and RF3 to produce the hybrid MS8 x RF3 does not result in interactions that cause compositional, agronomic and phenotypic changes that would raise safety concerns. Food and Feed Risk Assessment: Whole food feeding studies in broilers have not indicated any adverse health effects of oilseed rape MS8 x RF3. These studies also indicate that oilseed rape MS8 x RF3 is nutritionally equivalent to conventional oilseed rape. The PAT protein do not show sequence resemblance to other known toxins or IgE allergens, nor has PAT been reported to cause IgE mediated allergic reactions. Based on the current knowledge, the VKM GMO Panel concludes that oilseed rape MS8 x RF3 is nutritionally equivalent to conventional oilseed rape varieties, and that it is unlikely that the newly expressed proteins introduce a toxic or allergenic potential in food and feed derived from oilseed rape MS8 x RF3 compared to conventional oilseed rape. Environmental Risk Assessment: Considering the scope of the notification C/BE/96/01, excluding cultivation purposes, the environmental risk assessment is limited to exposure through accidental spillage of viable seeds of MS8, RF3 and MS8 x RF3 into the environment during transportation, storage, handling, processing and use of derived products. Oilseed rape is mainly a self-pollinating species, but has entomophilous flowers capable of both self- and cross-pollinating. Normally the level of outcrossing is about 30 %, but outcrossing frequencies up to 55 % are reported. Several plant species related to oilseed rape that are either cultivated, occurs as weeds of cultivated and disturbed lands, or grow outside cultivation areas to which gene introgression from oilseed rape could be of concern. These are found both in the Brassica species complex and in related genera. A series of controlled crosses between oilseed rape and related taxa have been reported in the scientific literature. Because of a mismatch in the chromosome numbers most hybrids have a severely reduced fertility. Exceptions are hybrids obtained from crosses between oilseed rape and wild turnip (B. rapa ssp. campestris) and to a lesser extent, mustard greens (B.juncea), where spontaneously hybridising and transgene introgression under field conditions have been confirmed. Wild turnip is native to Norway and a common weed in arable lowlands. Accidental spillage and loss of viable seeds of MS8, RF3 and MS8 x RF3 during transport, storage, handling in the environment and processing into derived products is likely to take place over time, and the establishment of small populations of oilseed rape MS8, RF3 and MS8 x RF3 cannot be excluded. Feral oilseed rape MS8, RF3 and MS8 x RF3 arising from spilled seed could theoretically pollinate conventional crop plants if the escaped populations are immediately adjacent to field crops, and shed seeds from cross-pollinated crop plants could emerge as GM volunteers in subsequent crops. However, both the occurrence of feral oilseed rape resulting from seed import spills and the introgression of genetic material from feral oilseed rape populations to wild populations are likely to be low in an import scenario in Norway. There is no evidence that the herbicide tolerant trait results in enhanced fitness, persistence or invasiveness of oilseed rape MS8, RF3 and MS8 x RF3, or hybridizing wild relatives, compared to conventional oilseed rape varieties, unless the plants are exposed to herbicides with the active substance glufosinate ammonium. Apart from the glufosinate tolerance trait, the resulting progeny will not possess a higher fitness and will not be different from progeny arising from cross-fertilisation with conventional oilseed rape varieties. Glufosinate ammonium-containing herbicides have been withdrawn from the Norwegian market since 2008, and the substance will be phased out in the EU in 2017 for reasons of reproductive toxicity. Overall Conclusion: Based on current knowledge, the VKM GMO Panel has not identified toxic, allergenic or altered nutritional properties of oilseed rape MS8, RF3 and MS8 x RF3 or its processed products compared to conventional oilseed rape. The VKM GMO Panel likewise concludes that oilseed rape MS8, RF3 and MS8 x RF3 are unlikely to have any adverse effect on the environment and agriculture in Norway in the context of its intended usage.

Immunogenicity of DNA vaccines can be increased by constructing the DNA in such a way that it encodes secreted homodimeric fusion proteins that target antigen-presenting cells (APCs). In this study, we have developed novel APC-targeting vaccine molecules with an increased flexibility due to introduction of a heterodimerization motif. The heterodimeric proteins permit four different fusions within a single molecule, thus allowing expression of two different APC-targeting moieties and two different antigens. Two types of heterodimeric fusion proteins were developed that employed either the ACID/BASE or the Barnase/Barstar motifs, respectively. The ACID/BASE heterodimeric vaccines conferred protection against challenges with either influenza virus or tumor cells in separate preclinical models. The ACID/BASE motif was flexible since a large number of different targeting moieties and antigens could be introduced with maintenance of specificity, antigenicity, and secretion. APC-targeting ACID/BASE vaccines expressing two different antigens induced antibody and T cell responses against either of the two antigens. Heterodimeric ACID/BASE DNA vaccines were of approximately the same potency as previously reported homodimeric DNA vaccines. The flexibility and potency of the ACID/BASE format suggest that it could be a useful platform for DNA vaccines that encode APC-targeting fusion proteins.

Small cationic guanyl-preferring ribonucleases (RNases) produced by the Bacillus species share a similar protein tertiary structure with a high degree of amino acid sequence conservation. However, they form dimers that differ in conformation and stability. Here, we have addressed the issues (1) whether the homologous RNases also have distinctions in catalytic activity towards different RNA substrates and interactions with the inhibitor protein barstar, and (2) whether these differences correlate with structural features of the proteins. Circular dichroism and dynamic light scattering assays revealed distinctions in the structures of homologous RNases. The activity levels of the RNases towards natural RNA substrates, as measured spectrometrically by acid-soluble hydrolysis products, were similar and decreased in the row high-polymeric RNA >>> transport RNA > double-stranded RNA. However, stopped flow kinetic studies on model RNA substrates containing the guanosine residue in a hairpin stem or a loop showed that the cleavage rates of these enzymes were different. Moreover, homologous RNases were inhibited by the barstar with diverse efficiency. Therefore, minor changes in structure elements of homologous proteins have a potential to significantly effect molecule stability and functional activities, such as catalysis or ligand binding.

The conventional methods of treating cancer with chemo- and radiotherapy present plenty of serious problems, such as low therapeutic index and high systemic toxicity. The advanced cancer treatment strategies utilize nanoformulations of drugs that can enter a tumor due to the enhanced permeability and retention (EPR) effect. However, EPR fails in the treatment of several human diseases. Mainstream biomedical studies are focused on creating the drugs that would enter the tumor with higher effectiveness and require smaller doses for administration. A two-stage drug delivery system is an encouraging alternative solution. At first, the primary, non-toxic targeting module is delivered to the tumor cells, followed by injection of the second complementary targeting module at a considerably lower dose, thus decreasing systemic toxicity. To meet the challenge, we have developed a two-stage drug delivery system (DDS), mediated by the high-affinity binding of the Barnase*Barstar protein pair. Barnase and Barstar act as lego bricks linking the first and the second modules on the surface of the cancer cell. Barnase (12 kDa) is a natural ribonuclease from Bacillus amyloliquefaciens, while Barstar (10 kDa) is its natural inhibitor. The Barnase*Barstar is one of the strongest known protein*protein complexes with Kaff = 1014 M−1 exhibiting extraordinarily stability in severe conditions. Artificial scaffold polypeptide DARPin9_29 genetically fused with Barstar served is a first module of the developed two-step DDS. DARPin9_29 (14 kDa) specifically recognizes the tumor marker HER2 overexpressed on human breast cancer cells. As a second module, a therapeutic nano-cargo was developed based on fluorescent polymer PLGA nanoparticles loaded with diagnostic Nile Blue dye and the chemotherapeutic drug doxorubicin. This nano-PLGA structure was covalently coupled to Barnase. We showed two-stage efficient labeling of HER2-overexpressing cancer cells using the first non-toxic module DARPin9_29-Barstar and the second toxic nano-module PLGA-Barnase. We demonstrated the doxorubicin-induced cytotoxicity of this two-step DDS based on polymer nanoparticles and proteinaceous Barnase-Barstar interface and showed more than 10-fold therapeutic dose reduction versus free doxorubicin. We believe that the developed two-step DDS based on PLGA nano-cargo and protein interface will promote the creation of new-generation cancer treatment strategies.

Sistemi za selekcijo bakterij, transformiranih z vektorskimi konstrukti, pogosto temeljijo na odpornosti proti antibiotikom. Zaradi nevarnosti prenosa genov za odpornost proti antibiotikom na druge bakterije ter splošno neželene uporabe antibiotikov, je potreba po uporabi varnejših, prehransko sprejemljivih selekcijskih sistemov velika. V tem magistrskem delu smo skušali vzpostaviti selekcijski sistem za bakterije Lactococcus lactis, ki temelji na sposobnosti presnove laktoze. Pripravili smo vektorski konstrukt pNZ8148_cat_lacF, iz katerega je z restrikcijskim encimom SalI mogoče enostavno izrezati obstoječi selekcijski označevalec, ki omogoča odpornost proti kloramfenikolu. V primerjavi z že obstoječim plazmidom za selekcijo na laktozi, pNZ8149, gen lacF na plazmidu pNZ8148_cat_lacF vsebuje lastni promotor in terminator transkripcije. Pri bakterijah L. lactis smo proučili tudi možnost uporabe potencialnega selekcijskega sistema toksin antitoksin, ki bi temeljil na ribonukleazi barnazi in njenem inhibitorju barstaru. Za ta namen smo pripravili vektorski konstrukt pNZDual_Ldh_Bar_Pnis_Bstar. Preverili smo, ali barnaza inhibira rast celic L. lactis in ali sočasno izražanje njenega inhibitorja barstara izniči ta vpliv, kar smo tudi potrdili. Gre za prvi poskus heterologne ekspresije barnaze in proteina barstar v vrsti L. lactis. The systems for selection of bacteria transformed with vector constructs are often based on antibiotic resistance. Due to the risk of transmission of antibiotic resistance genes to other bacteria and the generally undesirable use of antibiotics, there is the demand for safer, food grade selection systems. In this master's thesis, we attempted to set up a selection system for Lactococcus lactis, which would be based on the ability of bacterial cells to metabolize lactose. We prepared a vector construct pNZ8148_cat_lacF, which enables a simple removal of the existing chloramphenicol resistance selection marker, using the SalI restriction enzyme. Compared to the commercial plasmid pNZ8149, the lacF gene on plasmid pNZ8148_cat_lacF contains its own promoter and transcription terminator. We also examined the possibility of using a potential toxin antitoxin selection system based on ribonuclease barnase and its inhibitor barstar in L. lactis. For this purpose, we prepared a vector construct pNZDual_Ldh_Bar_Pnis_Bstar. We observed that barnase inhibits the growth of L. lactis and showed that the coexpression of barstar prevents the observed toxic activity of barnase. This has been the first attempt at heterologous expression of barnase and barstar proteins in bacterium L. lactis.

For precise ligation of a targeting and cytotoxic moiety, the use of Barnase-Barstar pair as a molecular glue is proposed for the first time. Targeting was mediated through the use of a scaffold protein DARPin_9–29 specific for the human epidermal receptor 2 (HER2) antigen that is highly expressed on some types of cancer and Barnase*Barstar native bacterial proteins interacted with each other with Kd 10−14 M. The approach proposed consists of prelabeling a target tumor with hybrid protein DARPin-Barnase prior to administration of cytotoxic component-loaded liposomes that have Barstar covalently attached to their surface. Based on in vivo bioimaging we have proven that DARPin-based Barnase*Barstar-mediated pretargeting possesses precise tumor-targeting capability as well as antitumor activity leading to apparent tumor-growth inhibition of primary tumors and distant metastases in experimental animals. The results obtained indicate that the new system combining DARPin and Barnase*Barstar can be useful both for the drug development and for monitoring the response to treatment in vivo in preclinical studies.

In many tissues and organisms, large amount of urea gets accumulated to maintain osmotic balance. To evade the threatening impact of urea, living organisms accumulate methylamines, a class of osmolytes, in proportion of 2:1 (urea:methylamine). To understand underlying cause(s) for protein-specific counteraction behavior, thermodynamic stability (ΔGDo) of three disulfide free proteins (myoglobin, bovine cytochrome c and barstar) in the mixture of urea and methylamine has been estimated from guanidinium chloride-(GdmCl) driven denaturation curves. Using the experimentally measured values of ΔGDo obtained in the presence of individual methylamines and urea, we predicted the molar ratio of urea and a methylamine required for perfect compensation for each of the proteins. Interestingly, for all proteins studied, a similar ratio has been observed for perfect compensation. The predicted ratio for perfect compensation in terms of thermodynamic parameters was about 2:1 M ratio of urea to methylamine. Furthermore, a partial counteraction was observed in the myoglobin and barstar. However, for bovine cytochrome c, perfect compensation was observed in both GdmCl- and heat-driven denaturations. Our observations clearly suggest that the counteraction phenomenon depends on the extent of the unfolding of the denatured states of proteins.

Surface modification of nanoparticles with various biologically active molecules makes it possible to realize the huge biomedical potential of various nanoobjects. Functionally active particles are usually obtained through stabilization by polymers and subsequent chemical conjugation with biomolecules, such as, antibodies or aptamers. This entails a number of problems, such as non-oriented conjugation, low coupling yield and the inability to easily vary the attached components on demand. To solve this problem, we developed a novel method of magnetite nanoparticles stabilization with simultaneous modification by functionally active protein – Barstar, for subsequent self-assembly of the necessary components with these particles through the interaction of high-affinity protein pair Barnase ∗ Barstar (Kaff = 1014 M−1). Namely, we developed a biocompatible Bs-C-Mms6 fusion protein containing the C-terminal part of the Mms6 (magnetite-binding protein of magnetotactic bacteria) and Barstar (an inhibitor of bacterial ribonuclease Barnase). We obtained stable in PBS magnetite nanoparticles modified with Bs-C-Mms6. These particles can be used for self-assembly with any type of Barnase-containing molecules. To demonstrate the effectiveness of this approach for the development of targeted nanoparticles, we performed a self-assembly of these particles with a fusion protein of Barnase and DARPin9.29, namely DARPin9.29-Bn. DARPin9.29 recognizes the extracellular domain of clinically important HER2/neu oncomarker. We have shown that such particles selectively bind this oncomarker and can be used to detect HER2/neu-positive cancer cells for diagnostic purposes.

The weighted ensemble (WE) strategy enables direct simulation of atomistic, fully-continuous protein–protein binding pathways in explicit solvent, yielding rigorous kinetics., A grand challenge in the field of biophysics has been the complete characterization of protein–protein binding processes at atomic resolution. This characterization requires the direct simulation of binding pathways starting from the initial, unbound state and proceeding through states that are too transient to be captured by experiment. Here, we applied the weighted ensemble path sampling strategy to orchestrate atomistic simulation of protein–protein binding pathways. Our simulation generated 203 fully-continuous and independent pathways along with rate constants for the binding process involving the barnase and barstar proteins. Results reveal multiple binding pathways along a “funnel-like” free energy landscape in which the formation of the “encounter complex” intermediate is rate-limiting followed by a relatively rapid rearrangement of the encounter complex to the bound state. Among all diffusional collisions, only ∼11% were productive. In the most probable binding pathways, the proteins rotated to a large extent (likely via electrostatic steering) in order to collide productively followed by “rolling” of the proteins along each other's binding interfaces to reach the bound state. Consistent with experiment, R59 was identified as the most kinetically important barnase residue for the binding process. Furthermore, protein desolvation occurs late in the binding process during the rearrangement of the encounter complex to the bound state. Notably, the positions of crystallographic water molecules that bridge hydrogen bonds between barnase and barstar are occupied in the bound-state ensemble. Our simulation was completed in a month using 1600 CPU cores at a time, demonstrating that it is now practical to carry out atomistic simulations of protein–protein binding.

Mms6 is a protein that plays crucial role in the biomineralization and formation of magnetosomes in magnetotactic bacteria Magnetospirillum magneticum (strain AMB-1). We developed a fusion protein of C-term part of Mms6 and Barstar (natural inhibitor of ribonuclease Barnase), namely, Bs-C-Mms6. This protein successfully stabilized uncoated monocrystalline Fe3O4 magnetite nanoparticles in buffered solutions. Here, we present data regarding the synthesis and characterization of magnetite nanoparticles stabilized with Bs-C-Mms6. For further interpretation of the data presented in this article, please see the research article ‘Self-assembling nanoparticles biofunctionalized with magnetite-binding protein for the targeted delivery to HER2/neu overexpressing cancer cells’ (Shipunova et al., 2018) [1] .

This work reports on modifying the Upstream Regulatory Module (URM, 1.5 Kb region upstream of the open reading frame) of Anther Expressing Gene 1 (AEG1) from cotton to achieve anther specific activity. AEG1 was identified in a previous study aimed to isolate a promoter with tapetum specific activity. Such a promoter could then be used to express barnase and barstar genes for developing male sterile and restorer lines for hybrid seed production in cotton. The AEG1 URM was observed to be active in tapetum as well as in roots making it unusable to drive the expression of barnase gene. Analysis of the URM showed the presence of several root specific motifs. Two modified AEG1 URMs were developed, by removing or mutating these motifs and its activity checked in tobacco. The activity of one of the modified URMs, AEG1(ΔBmut) was restricted to the anther tissue as observed using the reporter gene ß-glucuronidase. The study also demonstrates that male sterile lines could be developed in tobacco using the AEG1(ΔBmut) URM to express the barnase gene. This work thus shows the possibility of engineering promoters to achieve tissue specificity and to develop male sterile lines in cotton.

Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other non-degradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity—and, by extension, the myriad effects of ubiquitination on substrate proteins—arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrate’s C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Destabilization, however, appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a non-destabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single post-translational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality-control properties. (248 words)Significance StatementFluctuations on a protein energy landscapes encode the mechanistic basis for vital biological processes not always evident from static structures alone. Ubiquitination, a key posttranslational modification, can affect a protein’s energy landscape with consequences for proteasomal degradation, but the molecular mechanisms driving ubiquitin-induced energetic changes remain elusive. Here, we systematically characterize the energetic effects of ubiquitination at three sites on a model protein. We find that distinct thermodynamic mechanisms can produce the same outcome of ubiquitin-induced destabilization at sensitive sites. At a non-sensitive site, we observe formation of a substrate–ubiquitin interaction that may serve to protect against destabilization. This work will enable development of predictive models of the effect of ubiquitin at any given site on a protein with implications for understanding and engineering regulated ubiquitin signaling and protein quality control in vivo.

Plastid transformants form biofactories that are able to produce extra proteins in plastids when they are in a homoplasmic state. To date, plastid transformation has been reported in about twenty plant species; however, the production of homoplasmic plastid transformants is not always successful or easy. Heteroplasmic plants that contain wild-type plastids produce fewer target proteins and do not always successfully transfer transgenes to progeny. In order to promote the generation of homoplasmic plants, we developed a novel system using barnase-barster to eliminate wild-type plastids from heteroplasmic cells systematically. In this system, a chemically inducible cytotoxic barnase under a plastid transit signal was introduced into nuclear DNA and barster, which inhibits barnase, was integrated into plastid DNA with the primary selection markers aminoglycoside 3'-adenylyltransferase (aadA) and green fluorescence protein (GFP) gene. As expected, the expression of the plastid barnase was lethal to cells as seen in leaf segments, but barster expression in plastids rescued them. We then investigated the regeneration frequency of homoplasmic shoots from heteroplasmic leaf segments with or without barnase expression. The regeneration frequency of homoplasmic-like shoots expressing barnase-barster system was higher than that of shoots not expressing this. We expect that the application of this novel strategy for transformation of plastids will be supportive to generate homoplasmic plastid transformants in other plant species.

Anther and tapetum-specific genes are important for understanding male gametophyte development, as well as for their use in the development of barnase/barstar-gene based male sterility and restorer system for hybrid seed production. An essential component of the system is the availability of tapetum-specific promoters. In the present study, anther-specific genes were identified in cotton using microarray-based differential expression analysis, some of which show expression specific to the anthers at a stage where tapetum tissue was fully developed. Validation of the identified genes using RT-PCR and in situ hybridization identified one novel gene (AEG—Anther Expressing Gene) encoding a putative lipid binding protein as having a tapetum-specific expression. Further, three paralogs of the gene were identified in the cotton genome out of which the gene AEG1 (Anther Expressing Gene1) was found to express in the tapetum layer. Analysis of transgenic plants developed in cotton using 1.5 Kb promoter region of the of AEG1 gene fused upstream to the reporter gene β-glucuronidase revealed a broad window of expression of the AEG1 promoter in the tapetum tissue from the tetrad stage of anthers till the degeneration of the tapetum cells. Low levels of expression were also observed in the root tissues. Expression was not observed in the stem and leaves. The broad window of expression of AEG1 promoter in the tapetum tissue makes it a suitable candidate for the expression of the barstar gene for effective fertility restoration in the barnase/barstar system.

One important distinction between many tumor cell types and normal cells consists in the translocation of a number of intracellular proteins, in particular the 70 kDa heat shock protein (HSP70), to the surface of the plasma membrane. It has been demonstrated that such surface localization of HSP70 on tumor cells is recognized by cytotoxic effectors of the immune system, which increases their cytolytic activity. The mechanisms behind this interaction are not fully clear; however, the phenomenon of surface localization of HSP70 on cancer cells can be used to develop new approaches to antitumor immunotherapy. At the same time, it is known that the presence of HSP70 on a cells surface is not a universal feature of cancer cells. Many types of tumor tissues do not express membrane-associated HSP70, which limits the clinical potential of these approaches. In this context, targeted delivery of exogenous HSP70 to the surface of cancer cells with the aim of attracting and activating the cytotoxic effectors of the immune system can be considered a promising means of antitumor immunotherapy. Molecular constructs containing recombinant mini-antibodies specific to tumor-associated antigens (in particular, antibodies specific to HER2/neu-antigen and other markers highly expressed on the surface of a wide range of cancer cells) can be used to target the delivery of HSP70 to tumor tissues. In order to assess the feasibility and effectiveness of this approach, recombinant constructs containing a mini-antibody specific to the HER2/ neu-antigen in the first module and HSP70 molecule or a fragment of this protein in the second module were developed in this study. Strong selective interaction between the modules was ensured by a cohesive unit formed by the barnase:barstar pair, a heterodimer characterized by an unusually high constant of association. During testing of the developed constructs in in vitro models the constructs exhibited targeted binding to tumor cells expressing the HER2/neu antigen and the agents had a significant stimulating effect on the cytotoxic activity of NK cells against the respective cancer cells.

Nanoparticle surface engineering can change its chemical identity to enable surface coupling with functional biomolecules. However, common surface coupling methods such as physical adsorption or chemical conjugation often suffer from the low coupling yield, poorly controllable orientation of biomolecules, and steric hindrance during target binding. These issues limit the application scope of nanostructures for theranostics and personalized medicine. To address these shortfalls, we developed a rapid and versatile method of nanoparticle biomodification. The method is based on a SiO2-binding peptide that binds to the nanoparticle surface and a protein adaptor system, Barnase*Barstar protein pair, serving as a “molecular glue” between the peptide and the attached biomolecule. The biomodification procedure shortens to several minutes, preserves the orientation and functions of biomolecules, and enables control over the number and ratio of attached molecules. The capabilities of the proposed biomodification pla...

TA29 and A9 are genes from Nicotiana tabacum and Arabidopsis thaliana respectively, which express in a tapetum specific manner. The upstream regulatory modules (URMs; i.e. the promoter and the 5′UTR) of these genes have been used in development of male sterile and restorer lines expressing the barnase and barstar genes for hybrid seed production. While initial studies show that these URMs drive the expression in a tapetum specific manner, there are no recordings of unintended (leaky) expression driven by these URMs at ectopic locations due to position effect in developed transgenic lines. The information on leaky expression driven by tissue specific URMs is important for their use in developing transgenic plants. The present study records the leaky activity of both these URMs in transgenic tobacco lines using β-glucuronidase as a reporter gene. Leaky activity was observed in about one-fourth of the lines developed with TA29. Most interestingly in these lines, the leaky expression of the reporter gene was observed to be restricted to the meristematic tip region of the roots and at the leaf gap from where leaf trace diverges from stem bundles. Such a restricted and unique pattern of leaky activity of a tissue specific promoter or a URM has never been reported before, including the URM of the A9 gene analyzed in the present study. This observation suggests the presence of cryptic cis-elements within the URM of TA29 gene that can possibly activate it in meristematic tissue when integrated at certain ectopic locations. The URM of the A9 gene was also observed to show leaky activity. However, there was no unique pattern as observed with that of TA29. Further, in the study we also show that while the smaller (290 bp) length of TA29 URM can be used to drive the expression of barnase gene to develop male sterile lines, it adversely affects the regeneration of transgenic tobacco lines due to leaky expression. This adverse effect is significantly reduced when the full length (1.5 kb) URM of the TA29 gene is used.

The key to development of barnase-barstar transgene based hybrid seed technology is the availability of tightly regulated tapetum specific promoter, as any leaky expression of the barnase gene leads to several unintended effects. In the present study, we used two different tapetum specific promoters i.e. promoter of the RTS gene isolated from rice cultivar IR64 and the OsG6b promoter from japonica rice cultivar Hayayuki to express the barnase gene in rice transgenic lines. While viable male sterile transgenic lines could not be obtained with RTS promoter we could develop single copy male sterile lines when the barnase gene was expressed under the OsG6b promoter.

Canola is one of the most important plant oilseed crops. To avoid the threat of herbicides, the RF3 line with bar gene and barstar gene was developed, which can act as glufosinate resistance resources and restore fertility in hybrid lines. To assess the food safety of transgenic canola RF3, 2.5%, 5% and 10% GM canola RF3 and its non-GM isogenic line Drakkar were formulated into diet to feed Spragure-Dawley (SD) rats for 90 days. The effects on the general growth and toxicological parameters, as well as gut microbiota of rats, were evaluated. Several significant differences on body weight, feed consumption, relative organ weight, hematology and serum biochemistry were observed among rats in the 90-day feeding test. However, these statistical differences were randomly observed among different groups and were not dose-related, which were not considered to be biologically significant. Furthermore, the results of bacterial 16S rRNA sequencing of fecal samples showed that the diets containing GM canola did not disturb the balance of gut microbiota. In conclusion, the canola RF3 is considered as safe and wholesome as the non-GM canola based on this 90-day feeding test and gut microbiota analysis.