In the cell, proteins are synthesized from N to C terminus and begin to fold during translation. Cotranslational folding mechanisms are therefore linked to elongation rate, which varies as a function of synonymous codon usage. However, synonymous codon substitutions can affect many distinct cellular processes, which has complicated attempts to deconvolve the extent to which synonymous codon usage can promote or frustrate proper protein folding in vivo. Although previous studies have shown that some synonymous changes can lead to different final structures, other substitutions will likely be more subtle, perturbing predominantly the protein folding pathway without radically altering the final structure. Here we show that synonymous codon substitutions encoding a single essential enzyme lead to dramatically slower cell growth. These mutations do not prevent active enzyme formation; instead, they predominantly alter the protein folding mechanism, leading to enhanced degradation in vivo. These results support a model in which synonymous codon substitutions can impair cell fitness by significantly perturbing cotranslational protein folding mechanisms, despite the chaperoning provided by the cellular protein homeostasis network.

AbstractIn the cell, proteins are synthesized from N- to C-terminus and begin to fold during translation. Co-translational folding mechanisms are therefore linked to elongation rate, which varies as a function of synonymous codon usage. However, synonymous codon substitutions can affect many distinct cellular processes, which has complicated attempts to deconvolve the extent to which synonymous codon usage can promote or frustrate proper protein folding in vivo. Although previous studies have shown that some synonymous changes can lead to different final structures, other substitutions will likely be more subtle, perturbing predominantly the protein folding pathway without radically altering the final structure. Here we show that synonymous codon substitutions encoding a single essential enzyme lead to dramatically slower cell growth. These mutations do not prevent active enzyme formation; instead, they predominantly alter the protein folding mechanism, leading to enhanced degradation in vivo. These results support a model where synonymous codon substitutions can impair cell fitness by significantly perturbing co-translational protein folding mechanisms, despite the chaperoning provided by the cellular protein homeostasis network.SignificanceMany proteins that are incapable of refolding in vitro nevertheless fold efficiently to their native state in the cell. This suggests that more information than the amino acid sequence is required to properly fold these proteins. Here we show that synonymous mRNA mutations can alter a protein folding mechanism in vivo, leading to changes in cellular fitness. This work demonstrates that synonymous codon selection can play an important role in supporting efficient protein production in vivo.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding (CNB) domain in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides in the CNB domain to the pore opening. Surface plasmon resonance (SPR) is a powerful biophysical tool for quantitatively investigating ligand-protein and protein-protein interactions. Here we used SPR to detect ligand binding to the isolated C-linker/ CNB domain of HCN channels. The isolated C-linker/CNB domains of wildtype (WT) and L586W mutant HCN2 channels were immobilized on a NTA sensor chip and cAMP was injected over the protein coated surface. The mutant C-linker/CNB domain has been used before for ligand binding studies based on the changes in the fluorescence of the introduced tryptophan upon ligand binding. From the cyclic nucleotide concentration dependent SPR responses, we determined the binding affinity for cAMP to be 6.3 þ 2.6 mM for the WT and 10.8 þ 2.3mM for the mutant C-linker/CNB domains. The binding affinity determined for the mutant L586W C-linker-CNB domains determined with SPR is in agreement with the binding affinity of 13 þ 2 mM determined with the fluorescence-based method. These results indicate that SPR is well suited for the detection of binding of known HCN channel ligands. Therefore, SPR can be used to identify novel HCN channel regulators for treatment of diseases associated with abnormal functions of these channels, such as epilepsy and cardiac arrhythmias.