A change in the DNA sequence of a codon may not change the corresponding amino acid residue in the encoded protein because each residue can be encoded by several codons. This is called the Wobble hypothesis. Until now such synonymous changes in DNA were deemed “silent” mutations that did not affect the structure or function of the resulting protein.

Chemists at Pennsylvania State University have conducted computational and experimental studies to show this is not the case. “Silent” mutations, they claim, can still impact protein synthesis and function. Genetic changes that alter the speed of protein synthesis without altering the sequence of amino acids in the protein, can fold the protein in abnormal ways to change its activity, the team said. The findings highlight the importance of the rate of protein synthesis, in addition to sequence, in determining the structure and function of proteins.

“Our results provide an explanation and illustration of how kinetics can also control protein structure and function,” said Ed O’Brien, PhD, a professor of chemistry at Penn State, and the corresponding author of the study. “This has implications for any field involving protein synthesis. Protein misfolding also contributes to some human diseases. So, our work indicates an entirely new class of drug targets may exist for the development of future drugs.”

The study was published in the journal Nature Chemistry (“How synonymous mutations alter enzyme structure and function over long timescales”).

Using multiscale modeling of three Escherichia coli enzymes (type III chloramphenicol acetyltransferase, d-alanine–d-alanine ligase B, and dihydrofolate reductase) the researchers measured changes in specific activity due to synonymous mutations.

“We used to use ‘synonymous’ and ‘silent’ interchangeably to describe mutations that don’t change a protein’s sequence because it was thought that they wouldn’t alter the function of the protein,” said O’Brien. “But, we’ve known for some time now that not all synonymous mutations are silent. Over two decades ago, it was shown that synonymous mutations could reduce the activity of proteins, but it was still unknown what was happening at the molecular level to cause this change.”

“For a variety of reasons, some codons are translated at different speeds by the ribosome,” said Yang Jiang, PhD, assistant research professor of chemistry at Penn State and the first author of the paper. “For three different enzymes—specialized proteins that catalyze biochemical reactions—we simulated one version of the mRNA composed of fast translating codons and one version composed of slowly translating codons and then modeled the production of the nascent protein, how it is folded post-translationally, and its activity.”

Modeling study shows synonymous mutations that change the DNA sequence of a gene, but not the sequence of the encoded protein can impact protein production and function by changing the rate of protein synthesis. Top: Illustration of a new class of protein misfolding called a noncovalent lasso entanglement that can result from changes to the rate of protein synthesis caused by synonymous mutations. Bottom: Structure of a protein showing its native state and misfolded state with non-covalent lasso entanglement. [Yang Jiang, Penn State]

The researchers’ predictions for changes in protein activity based on simulation studies matched experimental results. They then examined the predicted protein structures and folding pathways from their models to identify molecular changes that could have led to the changes in activity. “In our models, we found a new class of protein misfolding that we call a ‘non-ovalent lasso entanglement,’” said Jiang. “Essentially, a portion of the protein forms a closed loop and one end of the protein incorrectly threads through the loop and gets trapped for long time periods.”

Such misfolding can reduce enzymatic activity by either occurring near the enzyme’s active site, or by avoiding recognition by chaperone proteins that normally identify and refold or remove misfolded proteins.

“We see inflection points during folding where the protein can either travel down a path that leads to a correctly folded protein or it can take a path that leads to the lasso entanglement. We call this ‘kinetic partitioning.’ How fast or slowly the protein is being translated—the kinetics of the process—seems to influence which path the protein is more likely to take,” said O’Brien.

These new findings on the effects of the rate of protein synthesis on its structure and function may affect studies in biochemistry, biotechnology, and medicine.