Intrinsically Disordered Regions Can Contribute Promiscuous Interactions to RNP Granule Assembly.

Eukaryotic cells contain large RNA-protein assemblies referred to as RNP granules, whose assembly is promoted by both traditional protein interactions and intrinsically disordered protein domains. Using RNP granules as an example, we provide evidence for an assembly mechanism of large cellular structures wherein specific protein-protein or protein-RNA interactions act together with promiscuous interactions of intrinsically disordered regions (IDRs). This synergistic assembly mechanism illuminates RNP granule assembly and explains why many components of RNP granules, and other large dynamic assemblies, contain IDRs linked to specific protein-protein or protein-RNA interaction modules.

Identification of NAD+ capped mRNAs in Saccharomyces cerevisiae.

RNAs besides tRNA and rRNA contain chemical modifications, including the recently described 5' nicotinamide-adenine dinucleotide (NAD) RNA in bacteria. Whether 5' NAD-RNA exists in eukaryotes remains unknown. We demonstrate that 5' NAD-RNA is found on subsets of nuclear and mitochondrial encoded mRNAs in Saccharomyces cerevisiae NAD-mRNA appears to be produced cotranscriptionally because NAD-RNA is also found on pre-mRNAs, and only on mitochondrial transcripts that are not 5' end processed.

A Derived Allosteric Switch Underlies the Evolution of Conditional Cooperativity between HOXA11 and FOXO1.

Transcription factors (TFs) play multiple roles in development. Given this multifunctionality, it has been assumed that TFs are evolutionarily highly constrained. Here, we investigate the molecular mechanisms for the origin of a derived functional interaction between two TFs, HOXA11 and FOXO1.

Computational design of protein-small molecule interfaces.

The computational design of proteins that bind small molecule ligands is one of the unsolved challenges in protein engineering. It is complicated by the relatively small size of the ligand which limits the number of intermolecular interactions. Furthermore, near-perfect geometries between interacting partners are required to achieve high binding affinities.

Exploring symmetry as an avenue to the computational design of large protein domains.

It has been demonstrated previously that symmetric, homodimeric proteins are energetically favored, which explains their abundance in nature. It has been proposed that such symmetric homodimers underwent gene duplication and fusion to evolve into protein topologies that have a symmetric arrangement of secondary structure elements--"symmetric superfolds". Here, the ROSETTA protein design software was used to computationally engineer a perfectly symmetric variant of imidazole glycerol phosphate synthase and its corresponding symmetric homodimer.

Computational design of an endo-1,4-beta-xylanase ligand binding site.

The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand.

Computational design of protein-ligand interfaces: potential in therapeutic development.

Computational design of protein-ligand interfaces finds optimal amino acid sequences within a small-molecule binding site of a protein for tight binding of a specific small molecule. It requires a search algorithm that can rapidly sample the vast sequence and conformational space, and a scoring function that can identify low energy designs. This review focuses on recent advances in computational design methods and their application to protein-small molecule binding sites.

A physical model for PDZ-domain/peptide interactions.

The PDZ domain is an interaction motif that recognizes and binds the C-terminal peptides of target proteins. PDZ domains are ubiquitous in nature and help assemble multiprotein complexes that control cellular organization and signaling cascades. We present an optimized energy function to predict the binding free energy (ΔΔG) of PDZ domain/peptide interactions computationally.

Human cardiac potassium channel DNA polymorphism modulates access to drug-binding site and causes drug resistance.

Expression of voltage-gated K channel, shaker-related subfamily, member 5 (KCNA5) underlies the human atrial ultra-rapid delayed rectifier K current (I(Kur)). The KCNA5 polymorphism resulting in P532L in the C terminus generates I(Kur) that is indistinguishable from wild type at baseline but strikingly resistant to drug block. In the present study, truncating the C terminus of KCNA5 generated a channel with wild-type drug sensitivity, which indicated that P532 is not a drug-binding site.

Calorimetric vs. van't Hoff binding enthalpies from isothermal titration calorimetry: Ba2+-crown ether complexation.

The 1:1 complexation reaction between Ba(2+) and 18-crown-6 ether is re-examined using isothermal titration calorimetry (ITC), with the goal of clarifying previously reported discrepancies between reaction enthalpies estimated directly (calorimetric) and indirectly, from the temperature dependence of the reaction equilibrium constant K (van't Hoff). The ITC thermograms are analyzed using three different non-linear fit models based on different assumptions about the data error: constant, proportional to the heat and proportional but correlated. The statistics of the fitting indicate a preference for the proportional error model, in agreement with expectations for the conditions of the experiment, where uncertainties in the delivered titrant volume should dominate.

Designing sequence to control protein function in an EF-hand protein.

The extent of conformational change that calcium binding induces in EF-hand proteins is a key biochemical property specifying Ca(2+) sensor versus signal modulator function. To understand how differences in amino acid sequence lead to differences in the response to Ca(2+) binding, comparative analyses of sequence and structures, combined with model building, were used to develop hypotheses about which amino acid residues control Ca(2+)-induced conformational changes. These results were used to generate a first design of calbindomodulin (CBM-1), a calbindin D(9k) re-engineered with 15 mutations to respond to Ca(2+) binding with a conformational change similar to that of calmodulin.

Engineering and design of ligand-induced conformational change in proteins.

The ability to manipulate ligand-induced conformational change, although representing a major challenge to the protein engineer, is an essential end point in efforts to produce novel functional proteins for biotechnology and therapeutic applications. Progress towards this goal requires determining not only what factors control the fold and stability of a protein, but also how ligand binding alters the complex conformational/energetic landscape. Important strides are being made on several fronts, including understanding the origin of long-range effects and allosteric structural mechanisms, using both experimental and theoretical approaches.