news & views on translation. Thus, the rate of translation of the polypeptide chain is not uniform along its length, but rather it proceeds in a pattern of fast and slow phases to coordinate synthesis and folding for optimal protein biogenesis5,6. Making use of the differing translation rates for synonymous codons, Clark and co-workers showed that altering the local rate of translation at specific regions of the protein could tune the folding of YKB, and amplify the preference for YK–B over Y–KB. By switching to rare codons in a region of the mRNA encoding the C-terminal (Blue) domain (Fig. 1c), translation in vivo produced more even more yellow fluorescence at the expense of blue fluorescence. This result indicates that the ribosome has slowed down at the rare codons, giving the Yellow half-domain even more time to associate with the central half-domain before the Blue halfdomain emerges. The effect on structure selection was tunable and correlated with codon rarity, with rarer codons producing proportionally more YK–B. Although global attenuation of translation speed can increase folding efficiency, here the change in codon usage did not significantly affect the overall translation rate of YKB. Although synonymous genetic substitutions have previously been found to alter local translation rate and folding efficiency, the coordination of multidomain protein folding and even the function of the final folded protein, Clark and co-workers have

presented the first study in which altered codon usage leads to predictable switching between two different native structures. These effects on protein folding can be visualized by considering the protein’s energy landscape, which can be viewed as a funnel whose width represents the conformational space available to the polypeptide chain, and whose depth represents its potential energy. Many proteins fold under thermodynamic control — that is, the native state is the structure at the bottom of the ‘folding funnel’ with lowest free energy. However, some proteins fold under kinetic control. In this case there are several funnels and thus several possible native structures, and the one that is selected depends on the conformations of the unfolded polypeptide and early intermediates, rather than the global minimum free energy. The team behind this study argue that co-translational folding may be especially important for proteins that fold under kinetic control, as the gradual emergence of the polypeptide chain from the ribosome restricts the conformational space available at the top of the funnel, effectively channelling the protein towards one or another of the possible folded structures. It is clear that elaborate mechanisms are in place to co-ordinate protein folding in vivo, and this work provides further insight into the subtleties controlling it. Such insights will help to provide foundations for the atomic resolution mapping of cotranslational folding pathways, the ultimate

goal in this field. By combining computer modelling and molecular dynamics simulations with innovative ways to transfer high-resolution experimental methods from the test tube into the cell7,8, we are acquiring the tools to achieve this aim. One could envisage extending the approach used by Clark and others so as to sequentially modulate translation rates at frequent points along the sequence to estimate the energies of intermediates and transition states. This would give further details of the in vivo folding landscape and the structures contained therein, and is analogous to the single-site amino acid substitutions (phi-value analysis) used so successfully to analyse folding in vitro. ❐ Elin M. Sivertsson and Laura S. Itzhaki are in the Department of Pharmacology at the University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK. e-mail: [email protected] References 1. Anfinsen, C. B. Science 181, 223–30 (1973). 2. Gershenson, A. & Gierasch, L. M. Curr. Opin. Struct. Biol. 21, 32–41 (2011). 3. Gloge, F., Becker, A. H., Kramer, G. & Bukau, B. Curr. Opin. Struct. Biol. 24, 24–33 (2014). 4. Sander, I. M., Chaney, J. L. & Clark, P. L. J. Am. Chem. Soc. 136, 858–61 (2014). 5. Zhang, G. & Ignatova, Z. Curr. Opin. Struct. Biol. 21, 25–31 (2011). 6. Pechmann, S., Willmund, F. & Frydman, J. Mol. Cell 49, 411–21 (2013). 7. Cabrita, L. D., Hsu, S. T., Launay, H., Dobson, C. M. & Christodoulou, J. Proc. Natl Acad. Sci. USA 106, 22239–44 (2009). 8. Ebbinghaus, S., Dhar, A., McDonald, J. D. & Gruebele, M. Nature Methods 7, 319–23 (2010).

SYNTHETIC BIOLOGY

Two-for-one designer labels

Labelling of proteins with pairs of fluorophores enables their conformations to be studied; however, complete incorporation of labels in multiple, pre-defined locations is very difficult. Now, a combination of double unnatural amino acid mutagenesis and selective chemical modification offers a general method to achieve this.

E. James Petersson and John B. Warner

U

nnatural amino acid mutagenesis — the swapping of a protein’s amino acids for unnatural analogues — could enable many important biochemical applications. One of the most exciting of these is the opportunity it offers for labelling proteins with fluorophore pairs for Förster resonance energy transfer (FRET) experiments. Such an approach forms an extremely powerful method for making real-time measurements of the distance between two sections of a protein in order to understand conformational

dynamics1,2. However, methods for site-specifically labelling proteins with multiple small fluorophores typically suffer from very low yields. As they report in Nature Chemistry, Jason Chin, long-time collaborator Ryan Mehl, and co-workers have developed a one-pot double labelling protocol that relies on the incorporation of two different unnatural amino acids that are then selectively modified with fluorophores3. Both the orthogonality of the modified translation system and the orthogonality of the subsequent chemical

NATURE CHEMISTRY | VOL 6 | MAY 2014 | www.nature.com/naturechemistry

© 2014 Macmillan Publishers Limited. All rights reserved

reactions are essential to the success of this elegant system. To insert even one unnatural amino acid into a protein expressed in living cells typically requires a mutant aminoacyl tRNA synthetase that can transfer an unnatural amino acid onto a tRNA molecule for incorporation into the protein at the ribosome, in a process known as charging 4. Crucially, this unnatural synthetase–tRNA pair must be orthogonal to the endogenous translation machinery, so that the mutant tRNA is not charged by existing synthetases 379

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