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Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol’s enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes.

Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol’s enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes.

o investigate the structural impact of phosphory-lation on the human histone H1.0 C-terminal domain,weperformed NMR structural studies of model peptides con-taining asingle phosphorylation site:T118-H1.0 (T118PKKmotif) andT140-H1.0 (T140PVK motif). Both model peptides aremainly disordered in aqueous solution in their non-phos-phorylated and phosphorylated forms, but become struc-tured in the presence of trifluoroethanol. The peptides T118-H1.0 and pT118-H1.0 contain two helical regions, along am-phipathic a helix spanning residues 104–115 and ashort a/310helix (residues 119–123),that are almostperpendicular inT118-H1.0 but have apoorly defined orientation in pT118-H1.0.Peptides T140-H1.0 and pT140-H1.0 form very similar a helicesbetween residues 141–147. The TPKK and TPVK motifs showthe same backbone conformation,but differ in their side-chain contacts;the Thr and pThr side chains interact withthe i + 2Lys side chain in the TPKK motif, andwith the i+ 3Lysside chain in the TPVK motif. The pT phosphate group inpT118-H1.0andpT140-H1.0haspKavaluesbelowtheintrinsicvalues, which can be explained by non-specific charge–chargeinteractions with nearbyLys. The non-polar ValintheTPVK motif accounts forthe pT140pKabeing closer to the in-trinsic pKavalue than the pT118pKa.Altogether,these resultsvalidate that minimalist strategies using model peptides canprovide structural details difficult to obtain in short-lived in-trinsically disordered proteins and domains.

Background: Employing methods to assess the quality of modeled protein structures is now standard practice in bioinformatics. In a broad sense, the techniques can be divided into methods relying on consensus prediction on the one hand, and single-model methods on the other. Consensus methods frequently perform very well when there is a clear consensus, but this is not always the case. In particular, they frequently fail in selecting the best possible model in the hard cases (lacking consensus) or in the easy cases where models are very similar. In contrast, single-model methods do not suffer from these drawbacks and could potentially be applied on any protein of interest to assess quality or as a scoring function for sampling-based refinement.Results: Here, we present a new single-model method, ProQ2, based on ideas from its predecessor, ProQ. ProQ2 is a model quality assessment algorithm that uses support vector machines to predict local as well as global quality of protein models. Improved performance is obtained by combining previously used features with updated structural and predicted features. The most important contribution can be attributed to the use of profile weighting of the residue specific features and the use features averaged over the whole model even though the prediction is still local.Conclusions: ProQ2 is significantly better than its predecessors at detecting high quality models, improving the sum of Z-scores for the selected first-ranked models by 20% and 32% compared to the second-best single-model method in CASP8 and CASP9, respectively. The absolute quality assessment of the models at both local and global level is also improved. The Pearson's correlation between the correct and local predicted score is improved from 0.59 to 0.70 on CASP8 and from 0.62 to 0.68 on CASP9; for global score to the correct GDT_TS from 0.75 to 0.80 and from 0.77 to 0.80 again compared to the second-best single methods in CASP8 and CASP9, respectively. ProQ2 is ava, QC 20131016

Receptors made visible: The described biotin-tagged small-molecule probes with excellent affinities for the CB 1 and CB 2 cannabinoid receptors (CB 1R and CB 2R) enable direct visualization of these receptors in native cellular systems, including neurons (see picture), microglia, and immune cells. This method could overcome some of the limitations of current methodologies and may help to dissect the complexity of the endogenous cannabinoid system. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A number of model structures of the CalA suggested by comparative modeling were tested by site-directed mutagenesis. Enzyme variants were created where amino acids predicted to play key roles for the lipase activity in the different models were replaced by an inert amino acid (alanine). The results from activity measurements of the overproduced and purified mutant enzymes indicate a structure where the active site consists of amino acid residues Ser184, His366, and Asp334 and in which there is no lid. This model can be used for future targeted modifications of the enzyme to obtain new substrate acceptance, better thermostability, and higher enantioselectivity.

We have developed a software suite to predict protein structures from sequence through the integration of multiple noncommercial programs. The Army and DoD medical and scientific communities will be able to use this software to annotate structures of sequenced pathogenic and host genomes. Such structural predictions can be used in therapeutic and vaccine design as well as many areas of basic biological research. In this work, initial assessments of the software are made. Most importantly, these tests include evaluation of the quality of predicted structural models as a function of sequence similarity to known protein structures. The presentation includes 16 briefing charts., Prepared in collaboration with the DoD Biotechnology High Performance Computing Software Applications Institute, Frederick, MD. See also ADM002075. Presented at the Army Science Conference (25th), "Transformational Army Science and Technology - Charting the future of S&T for the Soldier," held in Orlando, Florida on 27-30 Nov 2006. Published in proceedings of the same. The original document contains color images.

Fourteen 42-residue polypeptides have been designed to identify reactive sites for the catalysis of the decarboxylation of oxaloacetate, a chemical transformation that proceeds through the formation of an imine intermediate. The sequences fold into helix-loop-helix motifs and dimerise to four-helix bundles. The catalytically active lysine residues were incorporated in several surface exposed positions, but also in positions characterised by hydrophobic properties to reduce their pK(a) values. The molecular environments of the Lys residues were systematically varied, to find which residues were able to stabilise and bind the imine intermediate in the decarboxylation reaction. A two-residue Arg-Lys site formed the main component of the reactive site of the helix-loop-helix dimer Decarb-K34_R33, which obeyed saturation kinetics in catalysing the reaction with a k(cat)/K-M of 0.59m(-1) s(-1). The rate constant measured was nearly three orders of magnitude larger than the second-order rate constant of the butylamine-catalysed reaction (0.0011 M-1 s(-1)), and four orders of magnitude larger than the pseudo first-order rate constant of the uncatalysed reaction (1.3 x 10(-5) s(-1)). The sequence of Decarb-K34_R33 contained only a single lysine residue. It was flanked by an arginine in the preceding position in the sequence. A flanking Arg residue provided more efficient catalysis than a flanking Lys or Gln residue. Arginines in flanking positions in the helix, in positions four residues before or after the Lys in the sequence, are not as important in catalysis as the Arg of the Arg-Lys pair. The effect of pK(a) on the catalytic efficiency of the Lys residue in the decarboxylation reaction is well known. The identification of the role of the flanking Arg residue in catalysing decarboxylation, its optimal position, and the importance of conformational stability reported here sets the stage for developing a number of catalytic systems that depend on the formation of imine inte