A Computational Study of Cation−π Interactions vs Salt Bridges in Aqueous Media: Implications for Protein Engineering

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

From ab initio quantum mechanics to molecular neurobiology: A cation-pi binding site in the nicotinic receptor

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-pi 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-pi binding abilities and (ii) EC50 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 alpha 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 alpha 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 alpha 149 produces a constitutively active receptor

Site-specific incorporation of biotinylated amino acids to identify surface-exposed residues in integral membrane proteins

AbstractBackground: A key structural issue for all integral membrane proteins is the exposure of individual residues to the intracellular or extracellular media. This issue involves the basic transmembrane topology as well as more subtle variations in surface accessibility. Direct methods to evaluate the degree of exposure for residues in functional proteins expressed in living cells would be highly valuable. We sought to develop a new experimental method to determine highly surface-exposed residues, and thus transmembrane topology, of membrane proteins expressed in Xenopus oocytes.Results: We have used the in vivo nonsense suppression technique to incorporate biotinylated unnatural amino acids into functional ion channels expressed in Xenopus oocytes. Binding of 125I-streptavidin to biotinylated receptors was used to determine the surface exposure of individual amino acids. In particular, we studied the main immunogenic region of the nicotinic acetylcholine receptor. The biotin-containing amino acid biocytin was efficiently incorporated into five sites in the main immunogenic region and extracellular streptavidin bound to one residue in particular, α70. The position of α70 as highly exposed on the receptor surface was thus established.Conclusions: The in vivo nonsense suppression technique has been extended to provide the first in a potential series of methods to identify exposed residues and to assess their relative exposure in functional proteins expressed in Xenopus oocytes

A flow cytometry-based screen for synthetic riboswitches

Riboswitches regulate gene expression through direct, small molecule–mRNA interactions. The creation of new synthetic riboswitches from in vitro selected aptamers benefits from rapid, high-throughput methods for identifying switches capable of triggering dramatic changes in gene expression in the presence of a desired ligand. Here we present a flow cytometry-based screen for identifying synthetic riboswitches that induce robust increases in gene expression in the presence of theophylline. The performance characteristics of our newly identified riboswitches exceed those of previously described natural and synthetic riboswitches. Sequencing data and structure probing experiments reveal the ribosome binding site to be an important determinant of how well a switch performs and may provide insights into the design of new synthetic riboswitches

A Riboswitch-Based Inducible Gene Expression System for Mycobacteria

Research on the human pathogen Mycobacterium tuberculosis (Mtb) would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb

The formation of self-assembling helical aggregates driven by charge transfer interaction

Thesis (B.S.) in Chemistry--University of Illinois at Urbana-Champaign, 1994.Includes bibliographical references (leaves 30-31)U of I OnlyTheses restricted to UIUC community onl

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family of synthetic riboswitches adopts a kineti

Can Lone Pairs Bind to a π System? The Water···Hexafluorobenzene Interaction

Ab initio calculations reveal a significant binding interaction between water and hexafluorobenzene in a geometry that points the oxygen lone pairs directly into the face of the π system. The geometry is as anticipated from electrostatic arguments emphasizing the substantial quadrupole moment of the aromatic. A second, off-axis geometry is also found which is also consistent with a substantial electrostatic interaction

Cation-π interactions in structural biology

Cation-π interactions in protein structures are identified and evaluated by using an energy-based criterion for selecting significant sidechain pairs. Cation-π 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-π interaction. The sidechain of Arg is more likely than that of Lys to be in a cation-π 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-π interaction