Robert W. Newberry

n→π* interactions of amides and thioamides: implications for protein stability.

Carbonyl–carbonyl interactions between adjacent backbone amides have been implicated in the conformational stability of proteins. By combining experimental and computational approaches, we show that relevant amidic carbonyl groups associate through an n→π* donor–acceptor interaction with an energy of at least 0.27 kcal/mol. The n→π* interaction between two thioamides is 3-fold stronger than between two oxoamides due to increased overlap and reduced energy difference between the donor and acceptor orbitals. This result suggests that backbone thioamide incorporation could stabilize protein structures. Finally, we demonstrate that intimate carbonyl interactions are described more completely as donor–acceptor orbital interactions rather than dipole–dipole interactions.

Interplay of Hydrogen Bonds and n→π* Interactions in Proteins

Protein structures are stabilized by multiple weak interactions, including the hydrophobic effect, hydrogen bonds, electrostatic effects, and van der Waals interactions. Among these interactions, the hydrogen bond is distinct in having its origins in electron delocalization. Recently, another type of electron delocalization, the n→π* interaction between carbonyl groups, has been shown to play a role in stabilizing protein structure. Here we examine the interplay between hydrogen bonding and n→π* interactions. To address this issue, we used data available from high-resolution protein crystal structures to interrogate asparagine side-chain oxygen atoms that are both acceptors of a hydrogen bond and donors of an n→π* interaction. Then we employed natural bond orbital analysis to determine the relative energetic contributions of the hydrogen bonds and n→π* interactions in these systems. We found that an n→π* interaction is worth ~5-25% of a hydrogen bond and that stronger hydrogen bonds tend to attenuate or obscure n→π* interactions. Conversely, weaker hydrogen bonds correlate with stronger n→π* interactions and demixing of the orbitals occupied by the oxygen lone pairs. Thus, these two interactions conspire to stabilize local backbone-side-chain contacts, which argues for the inclusion of n→π* interactions in the inventory of non-covalent forces that contribute to protein stability and thus in force fields for biomolecular modeling.

n→π* Interactions Engender Chirality in Carbonyl Groups

An n→π* interaction stems from the delocalization of the electron pair (n) of a donor group into the antibonding orbital (π*) of a carbonyl group. Crystallographic analyses of five pairs of diastereoisomers demonstrate that an n→π* interaction can induce chirality in an otherwise planar, prochiral carbonyl group. Thus, a subtle delocalization of electrons can have stereochemical consequences.

A Key n→π* Interaction in N‑Acyl Homoserine Lactones

Many Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signal molecules for quorum sensing. The binding of AHLs to their target LuxR-type receptor proteins can effect changes in growth, virulence, and other phenotypes. LuxR-type receptors therefore present attractive pharmaceutical targets for control of bacterial pathogenesis. Here, we present X-ray crystallographic and computational evidence that the conformation of free AHLs is biased away from the conformation observed when bound to their cognate receptor due to the influence of an n→π* interaction. In this n→π* interaction, the p-type lone pair (n) of the N-acyl oxygen overlaps with the π* orbital of the lactone carbonyl group. This overlap results in the release of approximately 0.64 kcal/mol of energy. We also show that this interaction can be attenuated by installing electron-withdrawing groups on the N-acyl chain. Modulating this previously unappreciated interaction could present a new avenue toward effective inhibitors of bacterial quorum sensing.

The n→π* Interaction

The carbonyl group holds a prominent position in chemistry and biology not only because it allows diverse transformations but also because it supports key intermolecular interactions, including hydrogen bonding. More recently, carbonyl groups have been found to interact with a variety of nucleophiles, including other carbonyl groups, in what we have termed an n→π* interaction. In an n→π* interaction, a nucleophile donates lone-pair (n) electron density into the empty π* orbital of a nearby carbonyl group. Mixing of these orbitals releases energy, resulting in an attractive interaction. Hints of such interactions were evident in small-molecule crystal structures as early as the 1970s, but not until 2001 was the role of such interactions articulated clearly. These non-covalent interactions were first discovered during investigations into the thermostability of the proline-rich protein collagen, which achieves a robust structure despite a relatively low potential for hydrogen bonding. It was found that by modulating the distance between two carbonyl groups in the peptide backbone, one could alter the conformational preferences of a peptide bond to proline. Specifically, only the trans conformation of a peptide bond to proline allows for an attractive interaction with an adjacent carbonyl group, so when one increases the proximity of the two carbonyl groups, one enhances their interaction and promotes the trans conformation of the peptide bond, which increases the thermostability of collagen. More recently, attention has been paid to the nature of these interactions. Some have argued that rather than resulting from electron donation, carbonyl interactions are a particular example of dipolar interactions that are well-approximated by classical mechanics. However, experimental evidence has demonstrated otherwise. Numerous examples now exist where an increase in the dipole moment of a carbonyl group decreases the strength of its interactions with other carbonyl groups, demonstrating unequivocally that a dipolar mechanism is insufficient to describe these interactions. Rather, these interactions have important quantum-mechanical character that can be evaluated through careful experimental analysis and judicious use of computation. Although individual n→π* interactions are relatively weak (∼0.3-0.7 kcal/mol), the ubiquity of carbonyl groups across chemistry and biology gives the n→π* interaction broad impact. In particular, the n→π* interaction is likely to play an important role in dictating protein structure. Indeed, bioinformatics analysis suggests that approximately one-third of residues in folded proteins satisfy the geometric requirements to engage in an n→π* interaction, which is likely to be of particular importance for the α-helix. Other carbonyl-dense polymeric materials like polyesters and peptoids are also influenced by n→π* interactions, as are a variety of small molecules, some with particular medicinal importance. Research will continue to identify molecules whose conformation and activity are affected by the n→π* interaction and will clarify their specific contributions to the structures of biomacromolecules.

n→π* interactions in poly(lactic acid) suggest a role in protein folding

Poly(lactic acid) (PLA) is a versatile synthetic polyester. We noted that this depsipeptide analog of polyalanine has a helical structure that resembles a polyproline II helix. Using natural bond orbital analysis, we find that n→π* interactions between sequential ester carbonyl groups contribute 0.44 kcal mol(-1) per monomer to the conformational stability of PLA helices. We conclude that analogous n→π* interactions could direct the folding of a polypeptide chain into a polyproline II helix prior to the formation of hydrogen bonds between backbone amides.

A prevalent intraresidue hydrogen bond stabilizes proteins

Current limitations in de novo protein structure prediction and design suggest an incomplete understanding of the interactions that govern protein folding. Here we demonstrate that previously unappreciated hydrogen bonds occur within proteins between the amide proton and carbonyl oxygen of the same residue. Quantum calculations, infrared spectroscopy, and nuclear magnetic resonance spectroscopy show that these interactions share hallmark features of canonical hydrogen bonds. Biophysical analyses demonstrate that selective attenuation or enhancement of these C5 hydrogen bonds affects the stability of synthetic β-sheets. These interactions are common, affecting approximately 5% of all residues and 94% of proteins, and their cumulative impact provides several kcal/mol of conformational stability to a typical protein. C5 hydrogen bonds stabilize, especially, the flat β-sheets of the amyloid state, which is linked with Alzheimer’s disease and other neurodegenerative disorders. Inclusion of these interactions in computational force fields would improve models of protein folding, function, and dysfunction.

n→π* Interactions Are Competitive with Hydrogen Bonds

Because carbonyl groups can participate in both hydrogen bonds and n→π* interactions, these two interactions likely affect one another. Herein, enhancement of an amidic n→π* interaction is shown to reduce the ability of β-keto amides to tautomerize to the enol, indicating decreased hydrogen-bonding capacity of the amide carbonyl group. Thus, an n→π* interaction can have a significant effect on the strength of a hydrogen bond to the same carbonyl group.

4-Fluoroprolines: Conformational Analysis and Effects on the Stability and Folding of Peptides and Proteins

Proline is unique among proteinogenic amino acids because a pyrrolidine ring links its amino group to its side chain. This heterocycle constrains the conformations of the main chain and thus templates particular secondary structures. Proline residues undergo post-translational modification at the 4-position to yield 4-hydroxyproline, which is especially prevalent in collagen. Interest in characterizing the effects of this modification led to the use of 4-fluoroprolines to enhance inductive properties relative to the hydroxyl group of 4-hydroxyproline and to eliminate contributions from hydrogen bonding. The strong inductive effect of the fluoro group has three main consequences: enforcing a particular pucker upon the pyrrolidine ring, biasing the conformation of the preceding peptide bond, and accelerating cis/trans prolyl peptide bond isomerization. These subtle, yet reliable modulations make 4-fluoroproline-incorporation a complement to traditional genetic approaches for exploring structure-function relationships in peptides and proteins, as well as for endowing peptides and proteins with conformational stability.

The Main-Chain Oxygen: Unappreciated Effects on Peptide and Protein Structure

Current limitations in protein structure prediction and design suggest an incomplete understanding of the forces governing protein folding. As such, noncovalent interactions in proteins, particularly hydrogen bonds, have received great attention [1,2]. In common secondary structure patterns like the α-helix and β-sheet, main-chain N–H hydrogen bond donors approach their carbonyl acceptors approximately along the carbonyl bond axis [3], despite conventional wisdom that hydrogen bond energies are maximized when donors approach at 120° to the carbonyl bond axis [4]. This observation can be rationalized using a modern, quantummechanically based model of the carbonyl lone pairs that indicates that the two orbitals differ from the sp-hybridized VSEPR “rabbit ears” assumed commonly. Specifically, one lone pair, approximately sp-hybridized, is oriented along the carbonyl bond axis, while the second, purely p-orbital orients orthogonally (Figure 1). Canonical hydrogen bonds in protein secondary structure therefore often employ the s-rich lone pair; however, the role of the p-type lone pair is less clear. We have previously noted that backbone n→π* interactions are well poised to exploit this p-type lone pair [5]. In an n→π* interaction, the filled p-type lone pair of a carbonyl oxygen interacts with the empty π* orbital of an adjacent carbonyl group, and the mixing of these orbitals releases energy. These interactions have energies generally greater than 0.27 kcal/mol each [6], and are ubiquitous in folded proteins [7,8], particularly in the α-helix [9]. Yet, no analogous role for the p-type carbonyl lone pair has been identified in β-sheets. We now posit that a previously unappreciated hydrogen bond occurs within the backbone of individual residues in β-sheets.