Experimental demonstration of the benefits of somatic fusion and the consequences for allorecognition
Bastiaans, E. ; Debets, A.J.M. ; Aanen, D.K. - \ 2015
Evolution 69 (2015)4. - ISSN 0014-3820 - p. 1091 - 1099.
vegetative incompatibility - neurospora-crassa - heterokaryon incompatibility - natural-populations - filamentous fungi - recognition - evolution - selection - genetics - cooperation
Allorecognition, the ability to distinguish “self” from “nonself” based on allelic differences at allorecognition loci, is common in all domains of life. Allorecognition restricts the opportunities for social parasitism, and is therefore crucial for the evolution of cooperation. However, the maintenance of allorecognition diversity provides a paradox. If allorecognition is costly relative to cooperation, common alleles will be favored. Thus, the cost of allorecognition may reduce the genetic variation upon which allorecognition crucially relies, a prediction now known as “Crozier's paradox.” We establish the relative costs of allorecognition, and their consequences for the short-term evolution of recognition labels theoretically predicted by Crozier. We use fusion among colonies of the fungus Neurospora crassa, regulated by highly variable allorecognition genes, as an experimental model system. We demonstrate that fusion among colonies is mutually beneficial, relative to absence of fusion upon allorecognition. This benefit is due not only to absence of mutual antagonism, which occurs upon allorecognition, but also to an increase in colony size per se. We then experimentally demonstrate that the benefit of fusion selects against allorecognition diversity, as predicted by Crozier. We discuss what maintains allorecognition diversity
Immune activation mediated by the late blight resistance protein R1 requires nuclear localization of R1 and AVR1
Du, Y. ; Berg, J. ; Govers, F. ; Bouwmeester, K. - \ 2015
New Phytologist 207 (2015)3. - ISSN 0028-646X - p. 735 - 747.
disease-resistance - phytophthora-infestans - arabidopsis-thaliana - innate immunity - plant immunity - receptor - recognition - potato - gene - component
Resistance against oomycete pathogens is mainly governed by intracellular nucleotide-binding leucine-rich repeat (NLR) receptors that recognize matching avirulence (AVR) proteins from the pathogen, RXLR effectors that are delivered inside host cells. Detailed molecular understanding of how and where NLR proteins and RXLR effectors interact is essential to inform the deployment of durable resistance (R) genes. Fluorescent tags, nuclear localization signals (NLSs) and nuclear export signals (NESs) were exploited to determine the subcellular localization of the potato late blight protein R1 and the Phytophthora infestans RXLR effector AVR1, and to target these proteins to the nucleus or cytoplasm. Microscopic imaging revealed that both R1 and AVR1 occurred in the nucleus and cytoplasm, and were in close proximity. Transient expression of NLS- or NES-tagged R1 and AVR1 in Nicotiana benthamiana showed that activation of the R1-mediated hypersensitive response and resistance required localization of the R1/AVR1 pair in the nucleus. However, AVR1-mediated suppression of cell death in the absence of R1 was dependent on localization of AVR1 in the cytoplasm. Balanced nucleocytoplasmic partitioning of AVR1 seems to be a prerequisite. Our results show that R1-mediated immunity is activated inside the nucleus with AVR1 in close proximity and suggest that nucleocytoplasmic transport of R1 and AVR1 is tightly regulated.
Organizing Products with Complements versus Substitutes: Effects on Store Preferences as a Function of Effort and Assortment Perceptions
Diehl, K. ; Herpen, E. van; Lamberton, C. - \ 2015
Journal of Retailing 91 (2015)1. - ISSN 0022-4359 - p. 1 - 18.
consumer choice - price sensitivity - purchase - variety - behavior - too - organization - recognition - elasticity - strategies
Retailers often organize at least part of their assortment by displaying complementary products from different product categories together (e.g., a pair of pants with a shirt) rather than grouping items by product type (e.g., a pair of pants with other pants). However, little is known about how retailers should choose between complement-based and substitute-based organizations. The present paper shows that consumers’ preferences for such store organizations are a function of the effort and assortment perceptions cued by these organizational formats. Holding the underlying assortment constant, complement-based organizations are always more effortful than substitute-based organizations. This difference in effort can create downward pressure on complement-based store choice. Moreover, the effects of organization format on assortment perception depend on whether consumers hold a hedonic or utilitarian focus. When consumers have a highly hedonic focus, complement-based based stores create more positive assortment perceptions than substitute-based stores. Such positive assortment perceptions can, in turn, raise complement-based store choice. However, as consumers’ utilitarian focus increases, substitute-based assortments are seen as both easier and more attractive, leading to a strong advantage in store choice. Our findings provide actionable guidance for retailers considering various store organizations and suggest opportunities for future research.
The Rsm regulon of plant growth-promoting Pseudomonas fluorescens SS101: role of small RNAs in regulation of lipopeptide biosynthesis
Song, C. ; Voort, M. van der; Mortel, J. van de; Hassan, K.A. ; Elbourne, L.D.H. ; Paulsen, I.T. ; Loper, J.E. ; Raaijmakers, J.M. - \ 2015
Microbial Biotechnology 8 (2015)2. - ISSN 1751-7907 - p. 296 - 310.
signal-transduction pathway - escherichia-coli - phytophthora-infestans - bacterial genomes - soluble-rnas - fumarase c - aeruginosa - identification - cha0 - recognition
The rhizobacterium Pseudomonas fluorescens SS101 inhibits growth of oomycete and fungal pathogens, and induces resistance in plants against pathogens and insects. To unravel regulatory pathways of secondary metabolite production in SS101, we conducted a genome-wide search for sRNAs and performed transcriptomic analyses to identify genes associated with the Rsm (repressor of secondary metabolites) regulon. In silico analysis led to the identification of 16 putative sRNAs in the SS101 genome. In frame deletion of the sRNAs rsmY and rsmZ showed that the Rsm system regulates the biosynthesis of the lipopeptide massetolide A and involves the two repressor proteins RsmA and RsmE, with the LuxR-type transcriptional regulator MassAR as their most likely target. Transcriptome analyses of the rsmYZ mutant further revealed that genes associated with iron acquisition, motility and chemotaxis were significantly upregulated, whereas genes of the type VI secretion system were downregulated. Comparative transcriptomic analyses showed that most, but not all, of the genes controlled by RsmY/RsmZ are also controlled by the GacS/GacA two-component system. We conclude that the Rsm regulon of P.¿fluorescens SS101 plays a critical role in the regulation of lipopeptide biosynthesis and controls the expression of other genes involved in motility, competition and survival in the plant rhizosphere.
Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana
Du, J. ; Rietman, H. ; Vleeshouwers, V.G.A.A. - \ 2014
Journal of Visualized Experiments (2014)83. - ISSN 1940-087X - 7 p.
late blight resistance - mediated plant transformation - phytophthora-infestans - pathogen phytophthora - disease resistance - effector proteins - gene-expression - binary vector - agrobacterium - recognition
Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other's results.
Learning Apart Together: Towards an Integrated Competence Framework for Sustainable Entrepreneurship in Higher Education
Lans, T. ; Blok, V. ; Wesselink, R. - \ 2014
Journal of Cleaner Production 62 (2014). - ISSN 0959-6526 - p. 37 - 47.
opportunity-identification - venture performance - firm performance - management - perspective - growth - exploitation - capabilities - recognition - orientation
Sustainable entrepreneurs, i.e. those who proactively facilitate latent demands for sustainable development, are now in higher demand than ever before. Higher (business) education can play an important role in laying the foundation for these sustainable entrepreneurs. Traditionally, however, educational scholars focus either on the issue of education for sustainability or on entrepreneurship education. There is little work which explores and/or crosses the boundaries between these two disciplines, let alone work in which an effort is made to integrate these perspectives. In this article, a competence approach was taken as a first step to link the worlds of education for entrepreneurship and for sustainability because we postulate that both, apparently different, worlds can reinforce each other. Based on a literature review, focus group discussions with teachers in higher education (n = 8) and a structured questionnaire among students (n = 211), a set of clear, distinct competencies was developed, providing stepping stones for monitoring students' sustainable entrepreneurship development in school-based environments.
Is the reaction to chemical cues of predators affected by age or experience in fire salamanders (Salamandra salamandra)
Ibanez, A. ; Caspers, B.A. ; Lopez, P. ; Martin, J. ; Krause, E.T. - \ 2014
Amphibia-Reptilia 35 (2014)2. - ISSN 0173-5373 - p. 189 - 196.
diet cues - recognition - avoidance - risk - population - prospectus - preference - responses - tadpoles - behavior
Predation is one of the strongest forces driving natural selection. Predator success reduces future prey fitness to zero. Thus, recognition and avoidance of a potential predator is an essential fitness-relevant skill for prey. Being well equipped in the predator-prey arms race is highly adaptive. In this context we tested whether age and/or potential experience of fire salamanders (Salamandra salamandra) affected their behaviour towards the chemical signature of a potential predator. We evaluated the space use of salamanders in a test arena with a shelter containing chemical cues from a predator (i.e., a rat) and a clean shelter. Our results demonstrate that naïve subadult fire salamanders do show a significant behavioural reaction towards rat odour. However, they do not avoid the chemical cues of the potential predator, but instead have a significant preference for the shelter with rat faeces. In contrast to this, both the naïve adult and wild-caught adult fire salamanders showed neither a preference nor an avoidance of rat scent. These results could suggest a role of age in odour-based predator recognition in salamanders. Similarly, predator recognition through chemical cues could be more important early in life when the young fire salamanders are more vulnerable to predatory attacks and less important in other life stages when salamanders are less subjected to predation. In conclusion, future studies considering wild-caught subadults should disentangle the importance of previous experience for predator chemical recognition.
Mutational analysis of the Ve1 immune receptor that mediates Verticillium resistance in tomato
Zhang, Z. ; Song, Y. ; Liu, Chun-Ming ; Thomma, B.P.H.J. - \ 2014
PLoS ONE 9 (2014)6. - ISSN 1932-6203 - 8 p.
leucine-rich repeat - brassinosteroid perception - crystal-structure - structural basis - plant defense - arabidopsis - proteins - recognition - specificity - disease
Pathogenic Verticillium species are economically important plant pathogens that cause vascular wilt diseases in hundreds of plant species. The Ve1 gene of tomato confers resistance against race 1 strains of Verticillium dahliae and V. albo-atrum. Ve1 encodes an extracellular leucine-rich repeat (eLRR) receptor-like protein (RLP) that serves as a cell surface receptor for recognition of the recently identified secreted Verticillium effector Ave1. To investigate recognition of Ave1 by Ve1, alanine scanning was performed on the solvent exposed ß-strand/ß-turn residues across the eLRR domain of Ve1. In addition, alanine scanning was also employed to functionally characterize motifs that putatively mediate protein-protein interactions and endocytosis in the transmembrane domain and the cytoplasmic tail of the Ve1 protein. Functionality of the mutant proteins was assessed by screening for the occurrence of a hypersensitive response upon co-expression with Ave1 upon Agrobacterium tumefaciens-mediated transient expression (agroinfiltration). In order to confirm the agroinfiltration results, constructs encoding Ve1 mutants were transformed into Arabidopsis and the transgenes were challenged with race 1 Verticillium. Our analyses identified several regions of the Ve1 protein that are required for functionality.
The presence of a below-ground neighbour alters within-plant seed size distribution in Phaseolus vulgaris
Chen, B. ; During, H.J. ; Vermeulen, P.J. ; Anten, N.P.R. - \ 2014
Annals of Botany 114 (2014). - ISSN 0305-7364 - p. 937 - 943.
root competition - variable environments - optimal balance - number - recognition - germination - growth - consequences - adaptation - plasticity
* Background and Aims Considerable variation in seed size commonly exists within plants, and is believed to be favoured under natural selection. This study aims to examine the extent to which seed size distribution depends on the presence of competing neighbour plants. * Methods Phaseolus vulgaris plants rooting with or without a conspecific neighbourwere grown in soil with high or low nutrient availability. Seeds were harvested at the end of the growth cycle, the total nitrogen and phosphorus invested in seed production were measured and within-plant seed size distribution was quantified using a set of statistical descriptors. * Key Results Exposure to neighbours’ roots induced significant changes in seed size distribution. Plants produced proportionally more large seeds and fewer small ones, as reflected by significant increases in minimal seed size, mean seed size, skewness and Lorenz asymmetry coefficient. These effects were different from, and in several cases opposite to, the responses when the soil nutrient level was reduced, and were significant after correction for the amount of resources invested in seed production. * Conclusions Below-ground neighbour presence affects within-plant seed size distribution in P. vulgaris. This effect appears to be non-resource-mediated, i.e. to be independent of neighbour-induced effects on resource availability. It implies that, based on current environmental cues, plants can make an anticipatory adjustment of their investment strategy in offspring as an adaptation to the local environment in the future. Key words: Anticipatory maternal effect, bet-hedging, game theory, neighbour detection, Phaseolus vulgaris, kidney bean, root competition, seed-setting, seed size variation, size inequality, skewness. INTRODUCTION A considerable degree of variation in seed size within plants is commonly observed (Michaels et al., 1988; Silvertown, 1989; Ruiz de Clavijo, 2002; Vo¨ller et al., 2012). Such variation is often interpreted as an adaptive bet-hedging strategy (Harper et al., 1970; McGinley et al., 1987; McGinley and Charnov, 1988; Venable and Brown, 1988; Geritz, 1995). Many studies also reveal that plants modify the pattern of variation (i.e. distribution) to cope with their abiotic environmental conditions (e.g. temperature, Wulff, 1986; light, Galloway, 2001; nutrients, Galloway, 2001;water, Parciak, 2002). Herewe demonstrate that seed size distribution may also be modified in response to the presence of a below-ground neighbour. Within a species, seed size (following common practice, seed size refers to seedweight in this paper) often correlates positively with the competitiveness of the offspring (e.g. Houssard and Escarre´, 1991; Eriksson, 1999; Lehtila¨ and Ehrle´n, 2005; Dubois and Cheptou, 2012). Based on the trade-off, induced by resource limitation in plants, between competition (favours large seeds) and colonization (favours a large number of small seeds), Geritz (1995) extended an optimal offspring size model (Smith and Fretwell, 1974) by considering seedling competition and using
Degenerate target sites mediate rapid primed CRISPR adaptation
Fineran, P.C. ; Gerritzen, M.J.H. ; Suarez Diez, M. ; Künne, T.A. ; Boekhorst, J. ; Hijum, S.A.F.T. van; Staals, R.H.J. ; Brouns, S.J.J. - \ 2014
Proceedings of the National Academy of Sciences of the United States of America 111 (2014)16. - ISSN 0027-8424 - p. E1629 - E1638.
adaptive immune-systems - escherichia-coli - cas systems - streptococcus-thermophilus - bacterial immunity - defense system - foreign dna - dual-rna - recognition - interference
Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus, and the degradation of cognate invader DNA (protospacer). Invaders can escape type I-E CRISPR-Cas immunity in Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive-feedback process termed “priming.” Here, by using a randomized protospacer and PAM library and high-throughput plasmid loss assays, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6-nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of using degenerate target regions, with up to 13 mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position, and is nucleotide dependent. Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the coevolutionary arms race with their invaders.
Structural Basis for DNA Binding Specificity by the Auxin-Dependent ARF Transcription Factors
Boer, D.R. ; Freire Rios, A. ; Berg, W.A.M. van den; Saaki, T. ; Manfield, I.W. ; Kepinski, S. ; López-Vidrieo, I. ; Franco-Zorilla, J.M. ; Vries, S.C. de; Solano, R. ; Weijers, D. ; Coll, M. - \ 2014
Cell 156 (2014). - ISSN 0092-8674 - p. 577 - 589.
arabidopsis gene monopteros - response elements - vascular development - plant development - domains - family - embryo - embryogenesis - dimerization - recognition
Auxin regulates numerous plant developmental processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs), yet the mechanistic basis for generating specificity in auxin response is unknown. Here, we address this question by solving high-resolution crystal structures of the pivotal Arabidopsis developmental regulator ARF5/MONOPTEROS (MP), its divergent paralog ARF1, and a complex of ARF1 and a generic auxin response DNA element (AuxRE). We show that ARF DNA-binding domains also homodimerize to generate cooperative DNA binding, which is critical for in vivo ARF5/MP function. Strikingly, DNA-contacting residues are conserved between ARFs, and we discover that monomers have the same intrinsic specificity. ARF1 and ARF5 homodimers, however, differ in spacing tolerated between binding sites. Our data identify the DNA-binding domain as an ARF dimerization domain, suggest that ARF dimers bind complex sites as molecular calipers with ARF-specific spacing preference, and provide an atomic-scale mechanistic model for specificity in auxin response.
DNA-guided DNA interference by a prokaryotic Argonaute
Swarts, D.C. ; Jore, M.M. ; Westra, E.R. ; Zhu, Y. ; Janssen, J.H. ; Snijders, A.P. ; Wang, Y. ; Patel, D.J. ; Berenguer, J. ; Brouns, S.J.J. ; Oost, J. van der - \ 2014
Nature 507 (2014)7491. - ISSN 0028-0836 - p. 258 - 261.
thermophile thermus-thermophilus - silencing complex - structural basis - rna - archaebacteria - recognition - eubacteria - cleavage - sequence - protein
RNA interference is widely distributed in eukaryotes and has a variety of functions, including antiviral defence and gene regulation. All RNA interference pathways use small single-stranded RNA (ssRNA) molecules that guide proteins of the Argonaute (Ago) family to complementary ssRNA targets: RNA-guided RNA interference. The role of prokaryotic Ago variants has remained elusive, although bioinformatics analysis has suggested their involvement in host defence. Here we demonstrate that Ago of the bacterium Thermus thermophilus (TtAgo) acts as a barrier for the uptake and propagation of foreign DNA. In vivo, TtAgo is loaded with 5'-phosphorylated DNA guides, 13-25 nucleotides in length, that are mostly plasmid derived and have a strong bias for a 5'-end deoxycytidine. These small interfering DNAs guide TtAgo to cleave complementary DNA strands. Hence, despite structural homology to its eukaryotic counterparts, TtAgo functions in host defence by DNA-guided DNA interference
Transmission of fish-borne zoonotic trematodes (Heterophyidae) to common carp (Cyprinus carpio) is independent of density of fish and trematodes
Boerlage, A.S. ; Graat, E.A.M. ; Verreth, J.A.J. ; Jong, M.C.M. de - \ 2014
Journal of Helminthology 88 (2014)2. - ISSN 0022-149X - p. 183 - 188.
diplostomum-spathaceum - centrocestus formosanus - haplorchis pumilio - cercariae - host - infection - vietnam - recognition - populations - aquaculture
Fish-borne zoonotic trematodes (FZTs) can cause major human health problems. The aim of this study was to quantify the transmission of parapleurolophocercous cercariae to common carp (Cyprinus carpio) and to study the effect of the density of cercariae and the density of fish on transmission with respect to the volume of water and surface area of the bottom. Fish were kept individually either as controls (n= 91) or were exposed to 250 cercariae in tubes with a volume of 25, 50, 100, 250 or 500 ml water (n= 190) with a surface area of 4, 12, 21, 30 or 49 cm2 (n= 195). The dose to which the fish were exposed was kept constant. Infection occurred in 94–100% of fish, with a mean of 15–18 metacercariae per fish and the proportion of FZTs established at 0.06–0.07 metacercariae per cercariae per fish. Neither the prevalence of infection with FZTs nor the number of metacercariae per fish nor the proportion of FZTs established were significantly associated with differences in the density of cercariae or the density of fish per ml water or per cm2 surface area. Thus, it was concluded that the transmission of cercariae to fish is independent of density.
Predictive brain signals of linguistic development
Kooijman, V.M. ; Junge, C. ; Johnson, E.K. ; Hagoort, P. ; Cutler, A. - \ 2013
Frontiers in Psychology 4 (2013). - ISSN 1664-1078
event-related potentials - word segmentation - language-development - speech-perception - electrophysiological evidence - cerebral specialization - american infants - native-language - 1st year - recognition
The ability to extract word forms from continuous speech is a prerequisite for constructing a vocabulary and emerges in the first year of life. Electrophysiological (ERR) studies of speech segmentation by 9- to 12-month-old listeners in several languages have found a left-localized negativity linked to word onset as a marker of word detection. We report an ERR study showing significant evidence of speech segmentation in Dutch-learning 7-month-olds. In contrast to the left-localized negative effect reported with older infants, the observed overall mean effect had a positive polarity. Inspection of individual results revealed two participant sub-groups: a majority showing a positive-going response, and a minority showing the left negativity observed in older age groups. We retested participants at age three, on vocabulary comprehension and word and sentence production. On every test, children who at 7 months had shown the negativity associated with segmentation of words from speech outperformed those who had produced positive-going brain responses to the same input. The earlier that infants show the left-localized brain responses typically indicating detection of words in speech, the better their early childhood language skills.
LysM effectors: secreted proteins supporting fungal life
Kombrink, A. ; Thomma, B.P.H.J. - \ 2013
PLoS Pathogens 9 (2013)12. - ISSN 1553-7366 - 4 p.
chitin-triggered immunity - plant chitinases - virulence factor - pathogen - diversification - recognition - fragments - infection - receptor - binding
Structure and Activity of the RNA-Targeting Type III-B CRISPR-Cas Complex of Thermus thermophilus
Staals, R.H.J. ; Agari, Y. ; Maki-Yonekura, S. ; Zhu, Y. ; Taylor, D.W. ; Duijn, E. van; Barendregt, A. ; Vlot, M. ; Koehorst, J.J. ; Sakamoto, K. ; Masuda, A. ; Dohmae, N. ; Schaap, P.J. ; Doudna, J.A. ; Heck, A. ; Yonekura, K. ; Oost, J. van der; Shinkai, A. - \ 2013
Molecular Cell 52 (2013)1. - ISSN 1097-2765 - p. 135 - 145.
of-flight instrument - mass-spectrometry - escherichia-coli - silencing complex - antiviral defense - immune-system - protein - interference - transcription - recognition
The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes
Computational protein design with electrostatic focusing: experimental characterization of a conditionally folded helical domain with a reduced amino acid alphabet
Suarez Diez, M. ; Pujol, M. ; Matzapetakis, M. ; Jaramillo, A. ; Iranzo, O. - \ 2013
Biotechnology Journal 8 (2013)7. - ISSN 1860-6768 - p. 855 - 864.
solution nmr structure - structural basis - peptides - recognition - prediction - sequences - dynamics - energy - trifluoroethanol - optimization
Automated methodologies to design synthetic proteins from first principles use energy computations to estimate the ability of the sequences to adopt a targeted structure. This approach is still far from systematically producing native-like sequences, due, most likely, to inaccuracies when modeling the interactions between the protein and its aqueous environment. This is particularly challenging when engineering small protein domains (with less polar pair interactions than with the solvent). We have re-designed a three-helix bundle, domain B, using a fixed backbone and a four amino acid alphabet. We have enlarged the rotamer library with conformers that increase the weight of electrostatic interactions within the design process without altering the energy function used to compute the folding free energy. Our synthetic sequences show less than 15% similarity to any Swissprot sequence. We have characterized our sequences in different solvents using circular dichroism and nuclear magnetic resonance. The targeted structure achieved is dependent on the solvent used. This method can be readily extended to larger domains. Our method will be useful for the engineering of proteins that become active only in a given solvent and for designing proteins in the context of hydrophobic solvents, an important fraction of the situations in the cell
Picomolar inhibition of cholera toxin by a pentavalent ganglioside GM1os-calixarene
Garcia-Hartjes, J. ; Bernardi, S. ; Weijers, C.A.G.M. ; Wennekes, T. ; Gilbert, M. ; Sansone, F. ; Casnati, A. ; Zuilhof, H. - \ 2013
Organic & Biomolecular Chemistry 11 (2013). - ISSN 1477-0520 - p. 4340 - 4349.
heat-labile enterotoxin - gm1 mimics - binding - ligand - hexamethylenetetramine - oligosaccharide - recognition - affinity - crystal - design
Cholera toxin (CT), the causative agent of cholera, displays a pentavalent binding domain that targets the oligosaccharide of ganglioside GM1 (GM1os) on the periphery of human abdominal epithelial cells. Here, we report the first GM1os-based CT inhibitor that matches the valency of the CT binding domain (CTB). This pentavalent inhibitor contains five GM1os moieties linked to a calixarene scaffold. When evaluated by an inhibition assay, it achieved a picomolar inhibition potency (IC50 = 450 pM) for CTB. This represents a significant multivalency effect, with a relative inhibitory potency of 100000 compared to a monovalent GM1os derivative, making GM1os-calixarene one of the most potent known CTB inhibitors.
Editorial: The evolution of animal communication
Naguib, M. ; Price, J.J. - \ 2013
Behaviour 150 (2013)9-10. - ISSN 0005-7959 - p. 951 - 955.
Solvated protein-protein docking using Kyte-Doolittle-based water preferences
Kastritis, P.L. ; Visscher, K.M. ; Dijk, A.D.J. van; Bonvin, A.M.J.J. - \ 2013
Proteins : Structure, Function, and Bioinformatics 81 (2013)3. - ISSN 0887-3585 - p. 510 - 518.
ligand docking - biomolecular complexes - globular-proteins - drug design - molecules - recognition - interfaces - solvent - haddock - challenges
HADDOCK is one of the few docking programs that can explicitly account for water molecules in the docking process. Its solvated docking protocol starts from hydrated molecules and a fraction of the resulting interfacial waters is subsequently removed in a biased Monte Carlo procedure based on water-mediated contact probabilities. The latter were derived from an analysis of water contact frequencies from high-resolution crystal structures. Here, we introduce a simple water mediated amino acid - amino acid contact probability scale derived from the Kyte-Doolittle hydrophobicity scale and assess its performance on the largest high-resolution dataset developed to date for solvated docking. Both scales yield high-quality docking results. The novel and simple hydrophobicity scale, which should reflect better the physico-chemical principles underlying contact propensities, leads to a performance improvement of around 10% in ranking, cluster quality and water recovery at the interface compared to the statistics-based original solvated docking protocol.