Cation-interactions in protein structures are identified and evaluated by using an energy-based criterion for selecting significant sidechain pairs. Cation-interactions are found to be common among structures in the Protein Data Bank, and it is clearly demonstrated that, when a cationic sidechain (Lys or Arg) is near an aromatic sidechain (Phe, Tyr, or Trp), the geometry is biased toward one that would experience a favorable cation-interaction. The sidechain of Arg is more likely than that of Lys to be in a cation-interaction. Among the aromatics, a strong bias toward Trp is clear, such that over one-fourth of all tryptophans in the data bank experience an energetically significant cation-interaction.The three-dimensional structure of a protein is determined by a delicate balance of weak interactions. Hydrogen bonds, salt bridges, and the hydrophobic effect all play roles in folding a protein and establishing its final structure. In addition, the cation-interaction (1-3) is increasingly recognized as an important noncovalent binding interaction relevant to structural biology. Theoretical and experimental studies have shown that cation-interactions can be quite strong, both in the gas phase and in aqueous media. A number of studies have established a role for cation-interactions in biological recognition, especially in the binding of acetylcholine (4, 5). Here we present a detailed analysis of the extent and nature of cation-interactions that are intrinsic to a protein's structure and likely contribute to protein stability. We find that energetically significant cation-interactions are common in proteins-a ''typical'' protein will contain several. We also have documented some significant preferences for certain amino acid pairs as partners in a cation-interaction.Important early work indicated a role for cation-interactions in protein structures. Following work by Levitt and Perutz (6-8) suggesting a hydrogen bond between aromatic and amino groups, Burley and Petsko identified the ''amino aromatic'' interaction (9), in which NH-containing groups tend to be positioned near aromatic rings within proteins. It is now appreciated that the interaction of a cationic group with an aromatic-a cation-interaction-is much more favorable than an analogous interaction involving a neutral amine (10, 11). Important subsequent studies by Thornton (12-17) modified the Burley and Petsko analysis, especially with regard to the amino-aromatic ''hydrogen bond.'' In addition, explicit studies of Arg interacting with aromatic residues have been reported by Flocco and Mowbray (18) and by Thornton (14), and other efforts to search the Protein Data Bank (PDB) for cation-interactions between ligands and proteins have been reported (19,20).Previous protein database searches relied on geometric definitions of sidechain interactions, focusing on when a cationic sidechain displayed a certain distance͞angle relationship to an aromatic sidechain. The different geometries of Lys vs. Arg and Trp vs. Phe͞Tyr can make such comparisons proble...

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation-interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation-binding abilities and (ii) EC 50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues-tryptophan-149 of the ␣ subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of ␣ tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position ␣149 produces a constitutively active receptor.

A direct comparison of the energetic significance of a representative salt bridge vs a representative cation-π interaction in aqueous media and in a range of organic solvents is presented using ab initio electronic structures and the SM5.42R/HF solvation model of Cramer and Truhlar. The cation-π interaction shows a well depth of 5.5 kcal/mol in water, significantly larger than the 2.2 kcal/mol seen for the salt bridge. Consistent with this idea, a survey of the Protein Data Bank reveals that energetically significant cation-π interactions are rarely completely buried within proteins, but prefer to be exposed to solvent. These results suggest that engineering surface-exposed cation-π interactions could be a novel way to enhance protein stability.

We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-negative and Gram-positive bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria.

Riboswitches are RNA-based genetic control elements that regulate gene expression in a ligand-dependent fashion without the need for proteins. The ability to create synthetic riboswitches that control gene expression in response to any desired small-molecule ligand will enable the development of sensitive genetic screens that can detect the presence of small molecules, as well as designer genetic control elements to conditionally modulate cellular behavior. Herein, we present an automated high-throughput screening method that identifies synthetic riboswitches that display extremely low background levels of gene expression in the absence of the desired ligand and robust increases in expression in its presence. Mechanistic studies reveal how these riboswitches function and suggest design principles for creating new synthetic riboswitches. We anticipate that the screening method and design principles will be generally useful for creating functional synthetic riboswitches.

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Genetic selection provides the most powerful method to assay large libraries of biomolecules for function. However, harnessing the power of genetic selection for the detection of specific, nonendogenous small-molecule targets in vivo remains a significant challenge. The ability to genetically select for small molecules would provide a reaction-independent mechanism to clone biosynthesis genes from large DNA libraries and greatly facilitate the exploration of large libraries of mutant enzymes for improved synthetic capabilities including altered substrate specificities and enhanced regio- or stereoselectivities. While remarkable progress has been made in developing genetic methods to detect small molecules in vivo, many of these methods rely on engineering small-molecule-protein interactions which remains a difficult problem, and the potential for some of these systems to assay large libraries is limited by the low transformation efficiency and long doubling time of yeast relative to bacteria. Herein, we demonstrate that synthetic riboswitches that activate protein translation in response to a specific small molecule can be used to perform sensitive genetic screens and selections for the presence of small molecules in Escherichia coli. We further demonstrate that the exquisite molecular discrimination properties of aptamers selected in vitro translate directly into an in vivo genetic selection system. Finally, we demonstrate that a cell harboring a synthetic riboswitch with a particular ligand specificity can be selectively amplified from a million-fold larger pool of cells containing mutant riboswitches that respond to a closely related ligand, suggesting that it is possible to use genetic selection in E. coli to discover synthetic riboswitches with new ligand specificities from libraries of mutant riboswitches.

A major goal of synthetic biology is to reprogram cells to perform complex tasks. Here we show how a combination of in vitro and in vivo selection rapidly identifies a synthetic riboswitch that activates protein translation in response to the herbicide atrazine. We further demonstrate that this riboswitch can reprogram bacteria to migrate in the presence of atrazine. Finally, we show that incorporating a gene from an atrazine catabolic pathway allows these cells to seek and destroy atrazine.