earticle

영어

Protein engineers devoted lot of efforts to enhance the stability and biophysical properties of proteins through rational or irrational approaches. These approaches are limited by the restricted pool of 20 canonical amino acids. Recently, alternative approaches were developed to enrich and manipulate the functional and the biophysical properties of protein through incorporation of non-canonical amino acids (NCAA). For the purpose, genetic code engineering is the most frequently used methodology which reassigns the canonical amino acid of sense codon with NCAA. The advantage of this methodology is acquiring the synergistic effect through multiple site incorporation of NCAA can alter the biophysical properties of proteins. On the other hand, L-proline (Pro) plays a critical role in the protein structure by forming cis and trans peptidyl-proline bond conformation. The pyrrolidine ring of Pro structure adopts two alternative conformations as the Cγ-exo and Cγ- endo puckering. The replacement of proline with fluoroproline (FP) had been proven as a choice to tune or alter the biophysical properties of proteins. Fluorescent protein applications are widespread in the field of cell imaging as a model and reporter proteins. Among them DSRed have advantageous over other proteins, because of its emission and excitation in the red region of spectrum (longer wavelength) and exhibit less autofluorescence background in cellular imaging. The main disadvantage of DSRed is slow maturation and tendency to oligomerization. To circumvent this problem, protein engineers used directed evolution method for generating monomeric form DSRed variants with faster maturating property. Among them, mRFP1 is generated by introduction of 33 mutations into DSRed which showed ~10 times faster maturation speed. Here we used the genetic code engineering method to further enrich the stability and manipulate the biophysical properties of mRFP1by the incorporation of fluoroproline. A remarkable characteristic feature of this methodology is incorporation of NCAA to alter the protein properties without modifying the primary sequences. The global replacement of Pro residue with (4R)-FP into mRFP1 leads to fluorescent loss which was overcome by introducing canonical amino acid mutagenesis at Pro63 residue to Ala. Structural analysis of mRFP1 provide an insights into the key role of Pro63 for the red fluorescence emission by maintaining the planarity of the chromophore moiety. Here we showed ~2 fold enhanced thermal and chemical stability along with faster maturation of mRFP1 through a combination of canonical and non-canonical amino acid mutagenesis. Our study showed that a successful combination of canonical and non-canonical amino acid mutagenesis can enhance the protein biophysical property.