抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Through alternative processing of pre-messenger RNAs, individual mammalian genes often produce multiple mRNA and protein isoforms that may have related, distinct or even opposing functions. Here we report an in-depth analysis of 15 diverse human tissue and cell line transcriptomes on the basis of deep sequencing of complementary DNA fragments, yielding a digital inventory of gene and mRNA isoform expression. Analyses in which sequence reads are mapped to exon-exon junctions indicated that 92-94% of human genes undergo alternative splicing, 86% with a minor isoform frequency of 15% or more. Differences in isoform-specific read densities indicated that most alternative splicing and alternative cleavage and polyadenylation events vary between tissues, whereas variation between individuals was approximately twofold to threefold less common. Extreme or 'switch-like' regulation of splicing between tissues was associated with increased sequence conservation in regulatory regions and with generation of full-length open reading frames. Patterns of alternative splicing and alternative cleavage and polyadenylation were strongly correlated across tissues, suggesting coordinated regulation of these processes, and sequence conservation of a subset of known regulatory motifs in both alternative introns and 3' untranslated regions suggested common involvement of specific factors in tissue-level regulation of both splicing and polyadenylation.

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Alternative Isoform Regulation in Human Tissue Transcriptomes

New parameter sets of the GROMOS biomolecular force field, 54A7 and 54B7, are introduced. These parameter sets summarise some previously published force field modifications: The 53A6 helical propensities are corrected through new φ/ψ torsional angle terms and a modification of the N-H, C=O repulsion, a new atom type for a charged -CH(3) in the choline moiety is added, the Na(+) and Cl(-) ions are modified to reproduce the free energy of hydration, and additional improper torsional angle types for free energy calculations involving a chirality change are introduced. The new helical propensity modification is tested using the benchmark proteins hen egg-white lysozyme, fox1 RNA binding domain, chorismate mutase and the GCN4-p1 peptide. The stability of the proteins is improved in comparison with the 53A6 force field, and good agreement with a range of primary experimental data is obtained.

/pdf/definition-and-testing-of-the-gromos-force-field-versions-z7aeler3v7.pdf

Definition and testing of the GROMOS force-field versions 54A7 and 54B7

Iron Catalysis in Organic Synthesis

This review describes a multidimensional treatment of molecular recognition phenomena involving aromatic rings in chemical and biological systems. It summarizes new results reported since the appearance of an earlier review in 2003 in host-guest chemistry, biological affinity assays and biostructural analysis, data base mining in the Cambridge Structural Database (CSD) and the Protein Data Bank (PDB), and advanced computational studies. Topics addressed are arene-arene, perfluoroarene-arene, S⋅⋅⋅aromatic, cation-π, and anion-π interactions, as well as hydrogen bonding to π systems. The generated knowledge benefits, in particular, structure-based hit-to-lead development and lead optimization both in the pharmaceutical and in the crop protection industry. It equally facilitates the development of new advanced materials and supramolecular systems, and should inspire further utilization of interactions with aromatic rings to control the stereochemical outcome of synthetic transformations.

Aromatic rings in chemical and biological recognition: energetics and structures.

The development of catalytic systems for the controlled oxidation of C–H bonds remains a highly sought-after goal in chemistry owing to the great utility of such transformation toward expediting the synthesis and functionalization of organic molecules. Cytochrome P450 monooxygenases are the catalysts of choice in the biological world for mediating the oxidation of sp3 and sp2 C–H bonds with a high degree of chemo-, regio-, and stereoselectivity and in a wide array of compounds of varying complexity. The efficiency of these enzymes, compared with chemical methods, to catalyze the insertion of oxygen into unactivated C–H bonds under mild reaction conditions has sparked interest among researchers toward investigating and exploiting P450s for a variety of synthetic applications. Realizing the synthetic potential of these enzymes, however, depends upon the availability of effective strategies to tune the reactivity of natural P450s to obtain viable oxidation catalysts for the desired transformation. This revie...

Tuning P450 Enzymes as Oxidation Catalysts

The Fox-1 protein regulates alternative splicing of tissue-specific exons by binding to GCAUG elements. Here, we report the solution structure of the Fox-1 RNA binding domain (RBD) in complex with UGCAUGU. The last three nucleotides, UGU, are recognized in a canonical way by the four-stranded β-sheet of the RBD. In contrast, the first four nucleotides, UGCA, are bound by two loops of the protein in an unprecedented manner. Nucleotides U1, G2, and C3 are wrapped around a single phenylalanine, while G2 and A4 form a base-pair. This novel RNA binding site is independent from the β-sheet binding interface. Surface plasmon resonance analyses were used to quantify the energetic contributions of electrostatic and hydrogen bond interactions to complex formation and support our structural findings. These results demonstrate the unusual molecular mechanism of sequence-specific RNA recognition by Fox-1, which is exceptional in its high affinity for a defined but short sequence element.

/pdf/molecular-basis-of-rna-recognition-by-the-human-alternative-2n9h7dg3k5.pdf

Molecular basis of RNA recognition by the human alternative splicing factor Fox-1

Cytochrome P450 enzymes (P450s) are exceptional oxygenating catalysts with enormous potential in drug discovery, chemical synthesis, bioremediation, and biotechnology. Compared to their natural counterparts, however, engineered P450s often exhibit poor catalytic and cofactor coupling efficiencies. Obtaining native-like catalytic proficiencies is a mandatory first step towards utilizing the power of these versatile oxygenases in chemical synthesis. Cytochrome P450BM3 (119 kDa, B. megaterium) catalyzes the subterminal hydroxylation of long-chain (C12–C20) fatty acids. Its high activity and catalytic self-sufficiency (heme and diflavin reductase domains are fused in a single polypeptide chain) 5] make P450BM3 an excellent platform for biocatalysis. However, despite numerous reports of the heme domain being engineered to accept nonnative substrates, including short-chain fatty acids, aromatic compounds, alkanes, and alkenes, reports of preparative-scale applications of P450BM3 remain scarce. [9] P450BM3 function is finely regulated through conformational rearrangements in the heme and reductase domains and possibly also through hinged domain motions. 10] Hydroxylation of fatty acids occurs almost fully coupled to cofactor (NADPH) utilization (93–96% depending on the substrate). In the presence of nonnative substrates or when amino acid substitutions are introduced, the mechanisms controlling efficient catalysis in P450s are disrupted, leading to the formation of reactive oxygen species and rapid enzyme inactivation. High coupling efficiencies on substrates whose physicochemical properties are substantially different from the native substrates have not been achieved, and coupling efficiencies ranging from less than 1% to 30– 40% are typical. 8] Strategies for addressing this “coupling problem” are needed in order to take engineered P450s to larger-scale applications. Selective hydroxylation of short alkanes is a long-standing problem, for which no practical catalysts are available. In an effort to produce P450BM3-based biocatalysts for selective hydroxylation of small alkanes, we previously engineered this enzyme to accept propane and ethane (35E11 variant). Despite greater than 5000 total turnover (TTN) supported in vitro, the utility of this catalyst remained limited because of its poor in vivo performance (see below), which was mostly due to the low efficiencies for coupling the product formation to cofactor consumption (17.4% for propane and 0.01% for ethane oxidation). Our goal was to engineer a P450BM3 variant with nativelike activity and coupling efficiency towards a structurally challenging, nonnative substrate (propane) and evaluate the impact of these features on performance in preparative-scale biotransformations. To this end, we used a domain-based protein-engineering strategy, in which the heme, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) domains of the 35E11 variant were evolved separately in the context of the holoenzyme, and beneficial mutations were recombined in a final step (Figure 1). Previous work suggested that mutations in the reductase and linker regions can affect catalytic properties. However, no systematic engineering efforts had been undertaken to engineer the complete 1048 amino acid holoenzyme. Holoenzyme libraries outlined in Figure 1 were created using random, saturation, and site-directed mutagenesis and

Engineered Alkane‐Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties

Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions.

Using rational design, an engineered myoglobin-based catalyst capable of catalyzing the cyclopropanation of aryl-substituted olefins with catalytic proficiency (up to 46 800 turnovers) and excellent diastereo- and enantioselectivity (98–99.9 %) was developed. This transformation could be carried out in the presence of up to 20 g L−1 olefin substrate with no loss in diastereo- and/or enantioselectivity. Mutagenesis and mechanistic studies support a cyclopropanation mechanism mediated by an electrophilic, heme-bound carbene species and a model is provided to rationalize the stereopreference of the protein catalyst. This work shows that myoglobin constitutes a promising and robust scaffold for the development of biocatalysts with carbene-transfer reactivity.

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Rudi Fasan

Papers

Tuning P450 Enzymes as Oxidation Catalysts

Molecular basis of RNA recognition by the human alternative splicing factor Fox-1

Engineered Alkane‐Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties

Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions.

Highly diastereoselective and enantioselective olefin cyclopropanation using engineered myoglobin-based catalysts