Photo-antagonism of the GABAA receptor

Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation

Defining an Essence of Structure Determining Residue Contacts in Proteins

The network of native non-covalent residue contacts determines the three-dimensional structure of a protein. However, not all contacts are of equal structural significance, and little knowledge exists about a minimal, yet sufficient, subset required to define the global features of a protein. Characterisation of this “structural essence” has remained elusive so far: no algorithmic strategy has been devised to-date that could outperform a random selection in terms of 3D reconstruction accuracy (measured as the Ca RMSD). It is not only of theoretical interest (i.e., for design of advanced statistical potentials) to identify the number and nature of essential native contacts—such a subset of spatial constraints is very useful in a number of novel experimental methods (like EPR) which rely heavily on constraint-based protein modelling. To derive accurate three-dimensional models from distance constraints, we implemented a reconstruction pipeline using distance geometry. We selected a test-set of 12 protein structures from the four major SCOP fold classes and performed our reconstruction analysis. As a reference set, series of random subsets (ranging from 10% to 90% of native contacts) are generated for each protein, and the reconstruction accuracy is computed for each subset. We have developed a rational strategy, termed “cone-peeling” that combines sequence features and network descriptors to select minimal subsets that outperform the reference sets. We present, for the first time, a rational strategy to derive a structural essence of residue contacts and provide an estimate of the size of this minimal subset. Our algorithm computes sparse subsets capable of determining the tertiary structure at approximately 4.8 Å Ca RMSD with as little as 8% of the native contacts (Ca-Ca and Cb-Cb). At the same time, a randomly chosen subset of native contacts needs about twice as many contacts to reach the same level of accuracy. This “structural essence” opens new avenues in the fields of structure prediction, empirical potentials and docking

The concept of RNA-assisted protein folding: the role of tRNA

We suggest that tRNA actively participates in the transfer of 3D information from mRNA to peptides - in addition to its well-known, "classical" role of translating the 3-letter RNA codes into the one letter protein code. The tRNA molecule displays a series of thermodynamically favored configurations during translation, a movement which places the codon and coded amino acids in proximity to each other and make physical contact between some amino acids and their codons possible. This specific codon-amino acid interaction of some selected amino acids is necessary for the transfer of spatial information from mRNA to coded proteins, and is known as RNA-assisted protein folding

Protein 3D Structure Computed from Evolutionary Sequence Variation

The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing

First report of leaf spot on oil palm caused by Phyllosticta capitalensis in Malaysia

Oil palm (Elaeis guineensis Jacq.) is the most economically important crop in Malaysia and the world’s highest yielding oil crop. In March 2018, irregular spots with gray centers, dark brown edges, and chlorotic halos were observed on the leaves of oil palm trees of Tenera genotype planted at Block A, University Agriculture Park, Universiti Putra Malaysia, Malaysia. The area of production is 8 ha of conventional farming with temperature and humidity ranging from 33 to 34°C and 30 to 70%, respectively. There were a total of 1,192 oil palm trees in the area analyzed, and the disease incidence was estimated to be approximately 20%. Twelve leaf sections (5 mm2) of four diseased leaves from four different symptomatic oil palm trees were surface sterilized in 1% sodium hypochlorite for 2 min, rinsed twice with distilled water, dried on sterilized tissue paper, plated on potato dextrose agar (PDA) plates, and incubated at 27 ± 1°C for 14 days in the dark. A total of 12 single-spore isolates were obtained from all sampled leaf tissues. The single colonies of all fungal isolates were olivaceous greenish to olivaceous black with an irregular light olive margin on PDA. Conidia were obpyriform or nearly elliptic, unicellular, with a hyaline, unstable apical appendage, 5 to 12 µm long. The size of conidia was 10 to 15 μm in length and 6 to 8 μm in width. These morphological characters were consistent with the original description of Phyllosticta capitalensis (Glienke et al. 2011; Hennings 1908). For fungal identification to species level, the internal transcribed spacer region (ITS) of the representative isolate UPM-Ph1 was amplified utilizing the universal primers ITS5/ITS4 (White et al. 1990) and then sequenced. The 620-bp ITS sequence was deposited in GenBank under accession number MH699964. The phylogenetic analysis confirmed that the isolate UPM-Ph1 belonged to P. capitalensis, and it shares 100% homology with the ITS sequence of the reference pathogenic P. capitalensis strain CPC18884 (GenBank JF261465), which was isolated from the host Stanhopea graveolens in Brazil, as reported by Glienke et al. (2011). Pathogenicity testing was conducted by artificial inoculation of healthy oil palm (Tenera GH 500 series) leaves with isolate UPM-Ph1 under the conditions of 9-h day/15-h night cycle and using LED white light. Four leaves were wound inoculated in three parts with a sterile scalpel and pipetting a 10-µl droplet of a conidial suspension (106 conidia/ml) on each wound. Another four leaves were wound inoculated with sterilized distilled water and served as a control. All inoculated leaves and control leaves were incubated in a chamber with 98% relative humidity at 27 ± 1°C. After 7 days, typical lesions identical to those observed in the field appeared on the inoculated leaves but not on control leaves. P. capitalensis was successfully reisolated only from the inoculated leaves and identified by morphological observations. The pathogenicity test was repeated, and the same typical lesions were observed. P. capitalensis has been reported as a weak plant pathogen that causes various diseases in plants (Wikee et al. 2013a, 2013b). Unsuccessful reisolation of the fungus from the control leaves confirmed that the control plants were not already latently infected with the pathogen. To our knowledge, this is the first report of P. capitalensis causing leaf spot disease of oil palm in Malaysia. This pathogen is capable of affecting the health and yield of oil palm trees in the plantations substantially if no control measure is in place. Therefore, further investigation is needed to determine the distribution of P. capitalensis in oil palm plantations in Malaysia

Curr Opin Biotechnol

Novel high-throughput technologies for directed evolution enable experimental coverage of an impressive number of sequences. Nevertheless, the success of such experiments hinges on the initial sequence libraries. Here we consider the computational design of smart focused libraries and review insights from experimental strategies and theoretic advances in modelling their energy landscapes. In library design as in structure prediction, the applied energy function is the key. Current knowledge-based potentials have proven more successful than purely physics-based ones. Here we summarize novel approaches that extend the classical pairwise treatment of residue contacts towards adaptive knowledge-based multi-body potentials. We suggest that minimal sets of probabilistic constraints will lead to much more efficient sampling of permissible conformations and sequence space