Slow molecular recognition by RNA.

Molecular recognition is central to biological processes, function, and specificity. Proteins associate with ligands with a wide range of association rate constants, with maximal values matching the theoretical limit set by the rate of diffusional collision. As less is known about RNA association, we compiled association rate constants for all RNA/ligand complexes that we could find in the literature. Like proteins, RNAs exhibit a wide range of association rate constants. However, the fastest RNA association rates are considerably slower than those of the fastest protein associations and fall well below the diffusional limit. The apparently general observation of slow association with RNAs has implications for evolution and for modern-day biology. Our compilation highlights a quantitative molecular property that can contribute to biological understanding and underscores our need to develop a deeper physical understanding of molecular recognition events.

A Simple Thermoplastic Substrate Containing Hierarchical Silica Lamellae for High-Molecular-Weight DNA Extraction.

An inexpensive, magnetic thermoplastic nanomaterial is developed utilizing a hierarchical layering of micro- and nanoscale silica lamellae to create a high-surface-area and low-shear substrate capable of capturing vast amounts of ultrahigh-molecular-weight DNA. Extraction is performed via a simple 45 min process and is capable of achieving binding capacities up to 1 000 000 times greater than silica microparticles.

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction.

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5'-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5'-exon intermediate in self splicing to remain bound subsequent to 5'-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs.

Exploring purine N7 interactions via atomic mutagenesis: the group I ribozyme as a case study.

Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure-function relationships analogous to those seen for protein ligands and provided strong evidence for a well-formed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by ~20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes.

Evidence for an extended hydrogen bond network in the binding site of the nicotinic receptor: role of the vicinal disulfide of the alpha1 subunit.

The defining feature of the α subunits of the family of nicotinic acetylcholine receptors is a vicinal disulfide between Cys-192 and Cys-193. Although this structure has played a pivotal role in a number of pioneering studies of nicotinic receptors, its functional role in native receptors remains uncertain. Using mutant cycle analysis and unnatural residue mutagenesis, including backbone mutagenesis of the peptide bond of the vicinal disulfide, we have established the presence of a network of hydrogen bonds that extends from that peptide NH, across a ß turn to another backbone hydrogen bond, and then across the subunit interface to the side chain of a functionally important Asp residue in the non-α subunit. We propose that the role of the vicinal disulfide is to distort the ß turn and thereby properly position a backbone NH for intersubunit hydrogen bonding to the key Asp.

Structural rearrangements of the motor protein prestin revealed by fluorescence resonance energy transfer.

Prestin is a membrane protein expressed in the outer hair cells (OHCs) in the cochlea that is essential for hearing. This unique motor protein transduces a change in membrane potential into a considerable mechanical force, which leads to a cell length change in the OHC. The nonlinear capacitance in cells expressing prestin is recognized to reflect the voltage-dependent conformational change of prestin, of which its precise nature remains unknown. In the present work, we aimed to detect the conformational changes of prestin by a fluorescence resonance energy transfer (FRET)-based technique. We heterologously expressed prestin labeled with fluorophores at the COOH- or NH(2)-terminus in human embryonic kidney-293T cells, and monitored FRET changes on depolarization-inducing high KCl application. We detected a significant decrease in intersubunit FRET both between the COOH-termini and between the COOH- and NH(2)-termini. A similar FRET decrease was observed when membrane potential was directly and precisely controlled by simultaneous patch clamp. Changes in FRET were suppressed by either of two treatments known to abolish nonlinear capacitance, V499G/Y501H mutation and sodium salicylate. Our results are consistent with significant movements in the COOH-terminal domain of prestin upon change in membrane potential, providing the first dynamic information on its molecular rearrangements.

Probing the role of backbone hydrogen bonding in a critical beta sheet of the extracellular domain of a cys-loop receptor.

Probing the sheet: The network of hydrogen bonds formed in the outer beta sheet of the nicotinic acetylcholine receptor (nAChR; see figure) is fairly robust and tolerates single amide-to-ester mutations throughout. However, eliminating two proximal hydrogen bonds completely destroys receptor function; this adds further support to gating models that ascribe important roles to these beta strands of the nAChR extracellular domain.Long-range communication is essential for the function of members of the Cys-loop family of neurotransmitter-gated ion channels. The involvement of the peptide backbone in binding-induced conformational changes that lead to channel gating in these membrane proteins is an interesting, but unresolved issue. To probe the role of the peptide backbone, we incorporated a series of alpha-hydroxy acid analogues into the beta-sheet-rich extracellular domain of the muscle subtype of the nicotinic acetylcholine receptor, the prototypical Cys-loop receptor. Specifically, mutations were made in beta strands 7 and 10 of the alpha subunit. A number of single backbone mutations in this region were well tolerated. However, simultaneous introduction of two proximal backbone mutations led to surface-expressed, nonfunctional receptors. Together, these data suggest that while the receptor is remarkably robust in its ability to tolerate single amide-to-ester mutations throughout these beta strands, more substantial perturbations to this region have a profound effect on the protein. These results support a model in which backbone movements in the outer beta sheet are important for receptor function.

Long-range coupling in an allosteric receptor revealed by mutant cycle analysis.

The functional coupling of residues that are far apart in space is the quintessential property of allosteric proteins. For example, in Cys-loop receptors, the gating of an intrinsic ion channel is allosterically regulated by the binding of small molecule neurotransmitters 50-60 A from the channel gate. Some residues near the binding site must have as their primary function the communication of the binding event to the gating region. These gating pathway residues are essential to function, but their identification and characterization can be challenging. This work introduces a simple strategy, derived from mutant cycle analysis, for identifying gating pathway residues using macroscopic measurements alone. In the exemplar Cys-loop receptor, the nicotinic acetylcholine receptor, a well-characterized reporter mutation (betaL9'S) known to impact gating, was combined with mutations of target residues in the ligand-binding domain hypothesized or previously found to be functionally significant. A mutant cycle analysis of the macroscopic EC(50) measurements can then provide insights into the role of the target residue. This new method, elucidating long-range functional coupling in allosteric receptors, can be applied to several reporter mutations in a wide variety of receptors to identify previously characterized and novel mutations that impact the gating pathway. We support our interpretation of macroscopic data with single-channel studies. Elucidating long-range functional coupling in allosteric receptors should be broadly applicable to determining functional roles of residues in allosteric receptors.

An intersubunit hydrogen bond in the nicotinic acetylcholine receptor that contributes to channel gating.

The muscle nicotinic acetylcholine receptor is a large, allosteric, ligand-gated ion channel with the subunit composition alpha2betagammadelta. Although much is now known about the structure of the binding site, relatively little is understood about how the binding event is communicated to the channel gate, causing the pore to open. Here we identify a key hydrogen bond near the binding site that is involved in the gating pathway. Using mutant cycle analysis with the novel unnatural residue alpha-hydroxyserine, we find that the backbone N-H of alphaSer-191 in loop C makes a hydrogen bond to an anionic side chain of the complementary subunit upon agonist binding. However, the anionic partner is not the glutamate predicted by the crystal structures of the homologous acetylcholine-binding protein. Instead, the hydrogen-bonding partner is the extensively researched aspartate gammaAsp-174/deltaAsp-180, which had originally been identified as a key binding residue for cationic agonists.