The protein backbone plays a big role in protein stability. For example, hydrogen bonds between the NH of one amide and the carbonyl oxygen of another are involved in helices, sheets, and every other type of protein secondary structure.
Protein stability involves a variety of factors. In the physical chemical sense, it is defined as the difference in Gibbs free energy between the unfolded and the folded states. In some instances, it can also mean making proteins resistant to enzymes that want to degrade them. Stabilized proteins and peptides can be used as tools to better understand biological systems and as drugs to target diseases for which small-molecule drugs are ill suited.
But making changes that might stabilize the protein backbone have long been difficult. Unlike amino acid side chains, which can be changed by substituting another amino acid via straightforward biological means, simply swapping an amino acid typically does nothing to change a protein’s backbone. Instead, altering the backbone requires customized chemical syntheses.
In a symposium of the Division of Organic Chemistry at last month’s American Chemical Society national meeting in Philadelphia, chemists described several approaches for modifying the protein backbone. The modifications are making it possible to study the contribution of individual hydrogen bonds and other interactions to overall protein stability. In doing so, they are pointing to ways to design more stable peptides and proteins.
Replacing the amide bond (HNC=O) with electronically equivalent, or isosteric, groups—such as thioamides, olefins, and esters—is one way to stabilize the peptide backbone, as long as the substitution does not disturb other key interactions. Of these, thioamides are the only ones that are isosteres in the purest sense, but researchers in the field often also refer to the others as isosteres.
Ronald T. Raines, a biochemistry professor at the University of Wisconsin, Madison, described how a backbone stereoelectronic effect called the n to π-star (n→π*) interaction can be leveraged to stabilize proteins. For several years, he has championed the importance of this interaction, which involves the overlap of a lone electron pair on the carbonyl oxygen of one amino acid with the π antibonding orbital of the carbonyl on the neighboring amino acid.
“This interaction is strong and needs to be strong to maintain certain secondary structures in proteins, especially α-helices,” Raines told C&EN. “If you’re trying to make a protein and replace a peptide bond in an α-helical region with an isostere, you’d better choose one that can form good n→π* interactions. Otherwise, you’re going to pay a penalty.” Other interactions can compensate for the loss of a particular n→π* interaction, Raines said, but it could still cost 1 to 2 kcal. “Since proteins are typically stable by only 10 to 15 kcal/mol overall, that’s a big hit.”
All amino acids are involved in n→π* interactions, and some backbone substitutions preserve it better than others, Raines said. For example, olefins make poor n→π* acceptors, he said. Therefore substituting an olefin for a backbone amide is problematic when n→π* interactions play a large role in protein stability.
In contrast, thioamides, in which sulfur replaces the carbonyl oxygen (HNC=S), are excellent n→π* donors and acceptors, especially as a pair of neighboring thioamides. So peppering the protein backbone with thioamides is likely to boost protein stability, he noted. But he cautioned that sulfur’s size might make it difficult to use too many thioamides in a peptide or protein.
Other chemists are going beyond strictly isosteric backbone modifications to build more stable proteins. For example, W. Seth Horne, a chemistry professor at the University of Pittsburgh, incorporates flexible synthetic polymers and β-amino acids into folded proteins. β-Amino acids have an extra carbon in the backbone between the amine and the carbonyl carbon. Peptides containing such amino acids are more resistant to protein-degrading enzymes than are their natural α-amino acid-containing counterparts.
Horne’s group is developing a general set of rules for redesigning the protein backbone to mimic the various types of protein secondary structures. He and others have successfully mimicked α-helices using β-amino acids. In addition, Horne has mimicked exposed loops by substitution of the biocompatible polymer polyethylene glycol for the amino acids normally found in the sequence (ChemBioChem, DOI: 10.1002/cbic.201200200). He hopes that the various types of replacements can be modular and performed in tandem.
Horne uses the protein GB1, a 56-residue domain of streptococcal protein G, as a model system. This protein has a four-strand β-sheet, one α-helix, two tight turns, and two tightly packed loops on the surface. “We want to be able to replace the helix, the sheet, the loops, and the turns individually and then replace all of them together without having anything more than simple additive changes to stability,” Horne told C&EN. That’s the hope, but Horne acknowledges that synergistic effects—positive or negative—may occur when modifications are combined.
In yet another strategy to boost protein stability via backbone modifications, Paramjit S. Arora, a chemistry professor at New York University, is using covalent linkers to replace the backbone amide hydrogen bonds that normally hold α-helices together. He takes advantage of theory suggesting that once the first hydrogen bond forms, the rest of the helix zips into place. That first hydrogen bond involves an energy penalty of 4 to 5 kcal/mol. But after it forms, the other hydrogen bonds fall into place, giving back 0.5 kcal/mol per hydrogen bond on average.
Arora wants to “prepay” that first hydrogen bond penalty by incorporating a covalent linker between the first and fifth amino acids in a sequence. In addition, such linkers make the helix resistant to proteases.
Installing a covalent linker requires replacing two backbone atoms—the carbonyl oxygen of the first amino acid and the amide hydrogen of the fifth. The carbonyl oxygen is replaced with a carbon, resulting in a terminal alkene. The amide hydrogen is replaced with an allyl group. After the peptide containing the substitutions has been synthesized, Arora connects the first and fifth amino acids through ring-closing metathesis. Currently, such linkers can be added only to the amino terminus of the helix. The reaction does not work with internal alkenes, which are highly substituted and sterically hindered.
Arora plans to use such stabilized helices to disrupt interactions between proteins. “Protein-protein interactions are considered quite challenging to disrupt with small molecules because the interface is so large,” he told C&EN. “There’s a subset of protein-protein interactions which feature protein secondary structures at the interface.” He wants to disrupt such interactions by creating peptides that mimic the secondary structure in a way that is resistant to proteases. He hopes that such peptides could eventually be used as therapeutics. “Such targets are often not hit by small molecules with high specificity.”
Backbone-modified proteins are also useful biochemical tools. For example, E. James Petersson, symposium organizer and chemistry professor at the University of Pennsylvania, is using backbone modifications as probes of protein structure and folding. In particular, he has shown that thioamides can quench the fluorescence of the amino acids tryptophan and tyrosine or other fluorophores added to proteins. The quenching occurs only if the thioamide is within a particular distance, so it can be used to monitor how proteins fold.
At the meeting, Petersson described the semisynthesis of thioamide-labeled α-synuclein, an aggregation-prone protein involved in Parkinson’s disease. Because α-synuclein is too large to make via solid-phase synthesis, Petersson turned to a strategy called native chemical ligation. He expresses 130 of the protein’s 140 amino acids in Escherichia coli and connects that portion to a 10-residue peptide that includes a thioamide-labeled cysteine at the carboxy terminus. He can use fluorescence quenching to track the backbone conformation as the protein misfolds.
Meanwhile, Felicia A. Etzkorn, a chemistry professor at Virginia Polytechnic Institute & State University, is exploiting a stabilizing backbone modification to dissect cell cycle regulation.
She has for many years studied an isomerase called Pin1, which catalyzes the cis-trans isomerization of proline-containing peptides. Pin1 can do its job only if the cis-trans equilibrium of its substrate has been upset.
She has hypothesized that Cdc2, a kinase upstream of Pin1 in the cell cycle, is the enzyme responsible for upsetting that equilibrium. Cdc2’s substrate is Cdc25c, a proline-containing phosphatase. The phosphatase can be cis or trans, and its conformation when it is phosphorylated by Cdc2 wasn’t known.
That’s where the backbone modifications come in. Etzkorn used alkene isosteres of proline to determine which conformation of Cdc25c serves as Cdc2’s substrate. The olefin modification locked the proline conformation, and she found that Cdc2 exclusively uses trans-Cdc25c phosphatase as its substrate. She now thinks that the isomerization state of Cdc25c is important for regulating the cell cycle.
All of these approaches are giving scientists ways to explore the importance of the protein backbone and to make proteins with desirable properties. “The backbone is the part that is the same throughout the protein,” Petersson told C&EN. “Teasing apart exactly what it does is a task that lends itself to the classic physical organic approach of trying to tweak just one property at a time in a structure-function study. This is something you can only really do with synthetic chemistry.”