In Vivo N-Terminal Functionalization of Protein Figure 6. Equilibrium renaturation plots of GFPnt and GFPhs-r5M variants (fraction of recovered fluorescence). Urea-denatured protein samples were diluted in different concentrations of urea in refolding buffer and recovered fluorescence was normalized by dividing it by fluorescence of corresponding non-denatured samples diluted in similar fashion. Inlet table shows the aconcentration of urea at which the 50% of fluorescence is recovered during renaturation process under urea-unfolded conditions. doi:10.1371/journal.pone.0046741.g006 Discussion This study has demonstrated the biological N-terminal site- specific modification of GFP with bio-orthogonally reactive groups and its application to conjugation of the protein. The preparation of GFPs with N-terminal specific functional groups could be achieved by generating the internal Met-free GFP sequence that can be properly folded and subsequent use of the Met residue- specific substitution method. The produced unnatural GFPs were sufficiently homogeneous and showed almost comparable activity and folding efficiency to the natural GFP. Finally, the protein- protein conjugation using the functionalized GFPs was successfully demonstrated. Even though it was not done in this study, the N- terminally functionalized GFPs, GFPhs-r5M-Hpg or GFPhs-r5M- Aha, can be conjugated to the N-terminally functionalized scFvs, scFv-Aha or scFc-Hpg, which were prepared in our previous study [8], and the resulting GFP-scFv conjugates are fluorobodies which can be used for developing biosensors and diagnostics [32]. It is expected that the strategy employed in our study may not only enable the preparation of artificial N-terminal functionalized proteins on a large scale but also facilitate the various protein related bio-conjugation studies such as N-terminal specific glycosylation, PEGylation and labeling. Although in vivo site-specific incorporations of unnatural amino acids into target proteins using stop codons have been successfully achieved by suppressor based methodology [33], this approach has not been demonstrated for the N-terminus of proteins. The Met residue-specific methodology used in this study is probably the only method to install amino acid analogues in the N-terminus of a protein, and this approach is a handy choice for N-terminal specific functionalization of target proteins when the internal Met residues of the proteins can be changed into other ones. Sometimes the internal Met residues of proteins could be replaced with other hydrophobic amino acids such as Leu, Ile, and Ala [7,8]. However, it is easily expected that Met residues could play an important role in folding and stability of proteins, and these Met residues cannot be mutated to other ones without hurting the function or structure of the proteins. One of the key points in our study is that even the protein sequences such as GFP, of which the Met residues are very sensitive to mutations, could be engineered to be an internal Met-free sequence by introducing mutations known to enhance the folding efficiency and robustness of the protein. Although the mutations previously identified were used for the enhancement of GFP folding and stability in this study, current protein engineering approaches such as directed evolution and computational protein engineering can be efficiently em- ployed in the identification of such folding enhancement mutations for other proteins [24]. This implies that the generation of the internal Met-free sequences which can be properly folded may not be a serious problem anymore in the preparation of the N- terminal functionalized proteins through the in vivo Met-residue specific substitution method. This also indicates that it is possible to artificially manipulate the incorporation sites of target proteins by genetically reassigning the Met codons to any sites of the internal Met-free protein sequence, which would allow the selective site-specific functionalization of a protein. In the case that the unnatural amino acids incorporated into the first Met codon is not required, it can be removed by engineering the penultimate residue with non-bulky amino acids such as Gly, Ala, Cys [7,9,34]. There are some general or specific limitations in the proposed method, which should be considered before applying the method to bio-conjugations. For example, the method may be very PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e46741