Expanding Anfinsen’s Principle (Journal Club)

Paper: Expanding Anfinsen’s Principle: Contributions of Synonymous Codon Selection to Rational Protein Design.

In 1961, Anfinsen performed his now (in)famous denaturing experiment upon ribonuclease A, a small one hundred residue globular protein. He showed that it could both unfold and refold via the addition and subsequent removal of chemical substances. From this he concluded that a protein’s fold is that of its global free energy minimum and, consequently, all the information required to know the folded structure of a given protein is solely encoded within its sequence of amino acids. In 1972, Anfinsen was awarded the Nobel prize for this work from which stemmed the vast field of protein folding prediction, a global arms race to see who could best predict/find the elusive global minimum for any given protein.

Unfortunately, protein fold prediction is still in its infancy with regards to its predictive power. As a scientific community, we have made huge progress using homology models, whereby we use the structure of a protein with similar sequence to the one under investigation to provide a reasonable starting point for refinement. However, when there is no similar structure in existence, we struggle abysmally due to being forced to resort to de novo models. This lack of ability when we are given solely a sequence to work with, shows that that our fundamental understanding of the protein folding process must be incomplete.

An increasingly common viewpoint, one that is at odds with Anfinsen’s conclusions, is that there is additional information required for a protein to fold. One suggested source of information is in the production of the protein itself at the ribosome. Coined as cotranslational folding, it has been shown that a protein undergoing synthesis will fold as it emerges from the ribosome, not waiting until the entire sequence is synthesised. As such, the energy landscape that the protein must explore to fold is under constant change and development as more and more of the protein emerges from the ribosome. It is an iterative process of smaller searches as the energy landscape is modulated in steps to that of the complete amino acid sequence.

Another suggested source of information is within the degeneracy observed within the genetic code. Each amino acid is encoded for by up to 6 different codons, and as such, one can never determine exactly the coding DNA that created a given protein. While this degeneracy has been suggested as merely an effect to reduce the deleterious nature of point mutations, it has also been found that each of these codons are translated at a different rate. It is evident that information is consumed when RNA is converted into protein at the ribosome, sine reverse translation is impossible, and it is hypothesised that these variations in speed can alter the final protein structure.

Figure 1. Experimental design for kinetically controlled folding. (a) Schematic of YKB, which consists of three half-domains connected by flexible (AGQ)5 linkers (black lines). The Y (yellow) and B (blue) half-domains compete to form a mutually exclusive kinetically trapped folded domain with the central K (black) half-domain. The red wedge indicates the location of synonymous codon substitutions (see text). (b) Energy landscapes for proteins that fold under kinetic control have multiple deep minima, representing alternative folded structures, separated by large barriers. The conformations of the unfolded protein and early folding intermediates (colored arrows) determine the final folded state of the protein. Forces that constrict the unfolded ensemble (gray cone) can bias folding toward one structure. (c) During translation of the nascent chain by the ribosome (orange), folding cannot be initiated from the untranslated C-terminus, which restricts the ensemble of unfolded states and leads to the preferential formation of one folded structure.

Figure 1. Experimental design for kinetically controlled folding. (a) Schematic of YKB, which consists of three half-domains connected by flexible (AGQ)5 linkers (black lines). The Y (yellow) and B (blue) half-domains compete to form a mutually exclusive kinetically trapped folded domain with the central K (black) half-domain. The red wedge indicates the location of synonymous codon substitutions (see text). (b) Energy landscapes for proteins that fold under kinetic control have multiple deep minima, representing alternative folded structures, separated by large barriers. The conformations of the unfolded protein and early folding intermediates (colored arrows) determine the final folded state of the protein. Forces that constrict the unfolded ensemble (grey cone) can bias folding toward one structure. (c) During translation of the nascent chain by the ribosome (orange), folding cannot be initiated from the untranslated C-terminus, which restricts the ensemble of unfolded states and leads to the preferential formation of one folded structure. Image sourced from J. Am. Chem. Soc., 2014, 136(3),

The journal club paper by Sander et al. looked experimentally at whether both cotranslational folding and codon choice can have effect on the resultant protein structure. This was achieved through the construction of a toy model protein, consisting of three half domains as shown in Figure 1. Each of these half domains were sourced from bifluorescent proteins, a group of protein half domains that when combined fluoresce. The second half domain (K) could combine with either the first (Y) or the third (B) half domains to create a fluorophore, crucially this occurring in a non-reversible fashion such that once a full domain was formed it could not form the other. By choosing flurophores that differed in wavelength, it was simple to measure the ratio in which the species, YK-B or Y-KB, were formed.

They found that the ratio of these two species differed between in-vitro and in-vivo formation. When denatured Y-K-B species were allowed to refold, a racemic mixtrue was produced, both species found the be equally likely to form. In contrast, when synthesised at the ribosome, the protein showed an extreme bias to form the YK-B species as shown in Figure 2. They concluded that this is caused by cotranslational folding, the half domains Y and K having time to form the YK species before B was finished being produced. As pointed out by some members within the OPIG group, it would have been appreciated to see if similar results were produced if the species were reversed, such that B was synthesised first and Y last, but this point does not invalidate what was reported.

Figure 2. Translation alters YKB folded structure. (a) Fluorescence emission spectra of intact E. coli expressing the control fluorescent protein constructs YK (yellow) or KB (cyan). (b) Fluorescence emission spectra of intact E. coli expressing YKB constructs with common or rare codon usage (green versus red solid lines) versus the same YKB constructs folded in vitro upon dilution from a chemical denaturant (dashed lines). Numbers in parentheses correspond to synonymous codon usage; larger positive numbers correspond to more common codons. (c) E. coli MG1655 relative codon usage(3) for codons encoding three representative YKB synonymous mutants: (+65) (light green), (−54) (red), and (−100) (pink line).

Figure 2. Translation alters YKB folded structure. (a) Fluorescence emission spectra of intact E. coli expressing the control fluorescent protein constructs YK (yellow) or KB (cyan). (b) Fluorescence emission spectra of intact E. coli expressing YKB constructs with common or rare codon usage (green versus red solid lines) versus the same YKB constructs folded in vitro upon dilution from a chemical denaturant (dashed lines). Numbers in parentheses correspond to synonymous codon usage; larger positive numbers correspond to more common codons. (c) E. coli MG1655 relative codon usage(3) for codons encoding three representative YKB synonymous mutants: (+65) (light green), (−54) (red), and (−100) (pink line). Image sourced from J. Am. Chem. Soc., 2014, 136(3).

Following the above, they also probed the role of codon choice using this toy model system. They varied the codons choice over a small segment of residues between the K and B half domains, such that the had multitude of species which would be encoded either “faster” or “slower” across this region. Codon usage was used as the measure of speed, though this has yet to established within the literature as to its appropriateness. They found that the slower species increased the bias towards the YK-B over Y-KB, while faster species reduced it. This experiment shows clearly that codon choice has a role on a protein’s final structure, though they only show a large global effect. My work is primarily on whether codon choice has a role at the secondary structure level, so I will be avidly hoping that more experiments will follow that show the role of codons at finer levels.

In conclusion, Sander et al. performed one of the cleanest experimental proofs of cotranslational folding to date. Older evidence is more anecdotal in nature, with reports of protein X or Y changing in response to a single synonymous mutation. In contrast, the experiment reported here is systematic in the approach and leaves little room for doubt over the results. Secondly and more ground breaking, is the (again) systematic nature in which codon choice is investigated and shown to effect the global protein structure. This is one of those rare pieces of science which the conclusions are clear and forthcoming to all readers.

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