In Vivo N-Terminal Functionalization of Protein Figure 7. Protein-protein bio-conjugation of GFPhs-r5M-Hpg and GFPhs-r5M-Aha. (A) Copper (I)-catalyzed cycloaddition (CCCA) reaction between azide and alkyne incorporated to GFPhs-r5M resulted in the formation of triazole-linked protein-protein dimer bio-conjugation. (B) SDS- PAGE analysis of CCCA reaction between GFPhs-r5M proteins incorporated with Hpg (alkyne) and Aha (azide group). Lane 1: CCCA reaction without catalysis agents, CuSO4 and L-ascorbic acid; lane 2: CCCA reaction with catalysis agents, CuSO4 and L-ascorbic acid. This result shows the formation of triazole-linked protein-protein bio-conjugation of GFPhs-r5M dimer. M is molecular weight marker, thick arrow indicates the protein-protein conjugated GFPhs-r5M dimer of 55.2 kDa and grey arrow indicates the 27.6 kDa monomer of GFPhs-r5M containing Hpg and Aha respectively. doi:10.1371/journal.pone.0046741.g007 inefficient for the proteins with N-terminal signal sequences which can be cleaved in vivo or with hidden N-termini where the incorporated non-natural amino acids cannot be accessed once incorporated. In addition, the target proteins need to be purified to execute highly specific bio-conjugation reactions because the unnatural amino acids can also be slightly incorporated into endogenous proteins. In our study, the mutations of the Met residues in the buried hydrophobic core regions of GFP significantly lowered the folding efficiency of GFP, which was rescued by introducing the mutations for GFP folding enhancement, the majority of which were from the superfolder GFP [19]. According to the structural analysis of the superfolder GFP, the mutations resulted in the higher folding rate and folding robustness by inducing new noncovalent interactions involving ionized residues [19]. For instance, the S30R mutation contributed the formation of double salt bridges with E17 and E32 and intramolecular ionic network through four residues (E17, E32, R122 and E115) located in four different adjacent b-sheets in the structure. It is presumed that this kind of superfolder mutation effect compensated the destabilization effect caused by the mutations of the three Met residues in the hydrophobic-core [19]. The higher folding efficiency and folding robustness of GFPhs-r5M than those of GFPnt indicates that the superfolder mutations might presumably provide GFPnt-r5M with more stabilization energy than such compensating energy. On the other hand, we presume that the higher specific fluorescence of GFPhs-r5M than GFPnt might be caused by the mutations such as F64L, F99S and N149K mutations which can change the spectral properties of GFP by enhancing the hydrogen bonding networks around the chromophore [22,26,28,29]. Further mutagenesis and structural studies need to be performed to understand the improved folding and spectral properties of the variants more exactly. Supporting Information Figure S1 A. SDS-PAGE analysis of the soluble and insoluble protein fractions of GFPnt and GFPnt-r2M. (M: molecular weight marker, lane 1: insoluble fraction of GFPnt, lane 2: soluble fraction of GFPnt, lane 3: soluble fraction of GFPhs-r2M, lane 4: insoluble fraction of GFPhs-r2M) B. SDS-PAGE analysis of the soluble and insoluble protein fractions of GFPnt-r3M. (S, soluble fraction; I, insoluble fraction). C. SDS-PAGE analysis of the soluble and insoluble protein fractions of GFPnt-r5M. (S, soluble fraction; I, insoluble fraction). D. SDS-PAGE analysis of the soluble and insoluble protein fractions of GFPhs-r5M and GFPnt. (S, soluble fraction; I, insoluble fraction; M, molecular weight marker). The expected size of 27.6 kDa is indicated by arrow in the figures. (TIF) Figure S2 Chemical structure of natural L-methionine (Met) and their unnatural surrogates L-homopropargyl- glycine (Hpg) and L-azidohomoalanine (Aha) (Mol. Wt: molecular weight). (TIF) Figure S3 ESI-MS analysis of GFPhs-r5M incorporated with Hpg and Aha. GFPhs-r5M (A), GFPhs-r5M-Hpg (B) PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e46741