Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position.

In order to alter the fluorescence properties of green fluorescent protein (GFP), aromatic non-natural amino acids were introduced into the Tyr66 position of GFP in a cell-free translation system using a four-base codon method. Two non-natural mutants (O-methyltyrosine and p-aminophenylalanine mutants) out of 18 mutants showed blue-shifted but weak fluorescence compared with wild-type GFP. Then the aminophenylalanine mutant was sequence optimized by introducing random mutations around the Tyr66 site. For this purpose, a method for random mutation of non-natural proteins in a cell-free system was developed. Three aminophenylalanine mutants with Y145F, Y145L and Y145 M mutations were obtained, which exhibited increased fluorescence by 1.5-, 3- and 4-fold, respectively. These results indicate that random mutation around non-natural amino acids is useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here will be applicable to other non-natural mutant proteins in a high-throughput manner.

[1]  S J Remington,et al.  Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[2]  M. Sisido,et al.  Site-Directed Incorporation of p-Nitrophenylalanine into Streptavidin and Site-to-Site Photoinduced Electron Transfer from a Pyrenyl Group to a Nitrophenyl Group on the Protein Framework , 1998 .

[3]  M. Sisido,et al.  Position‐specific incorporation of dansylated non‐natural amino acids into streptavidin by using a four‐base codon , 2004, FEBS letters.

[4]  S. Hecht,et al.  Structurally Modified Firefly Luciferase. Effects of Amino Acid Substitution at Position 286 , 1997 .

[5]  R Y Tsien,et al.  Understanding, improving and using green fluorescent proteins. , 1995, Trends in biochemical sciences.

[6]  S J Remington,et al.  Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein. , 1998, Structure.

[7]  M. Saraste,et al.  FEBS Lett , 2000 .

[8]  M. Sisido,et al.  Incorporation of Nonnatural Amino Acids into Streptavidin through In Vitro Frame-Shift Suppression , 1996 .

[9]  Roger Y. Tsien,et al.  Crystal Structure of the Aequorea victoria Green Fluorescent Protein , 1996, Science.

[10]  Peter G. Schultz,et al.  A New Strategy for the Synthesis of Glycoproteins , 2004, Science.

[11]  S. Boxer,et al.  Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Sisido,et al.  Efficient Incorporation of Nonnatural Amino Acids with Large Aromatic Groups into Streptavidin in In Vitro Protein Synthesizing Systems , 1999 .

[13]  H. Lester,et al.  Unnatural amino acid mutagenesis in mapping ion channel function , 2003, Current Opinion in Neurobiology.

[14]  Roger Y. Tsien,et al.  Improved green fluorescence , 1995, Nature.

[15]  R Y Tsien,et al.  Wavelength mutations and posttranslational autoxidation of green fluorescent protein. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[16]  P G Schultz,et al.  A general method for site-specific incorporation of unnatural amino acids into proteins. , 1989, Science.

[17]  M. Sisido,et al.  Photoswitching of peroxidase activity by position‐specific incorporation of a photoisomerizable non‐natural amino acid into horseradish peroxidase , 2002, FEBS letters.

[18]  M. Sisido,et al.  Incorporation of nonnatural amino acids into proteins by using various four-base codons in an Escherichia coli in vitro translation system. , 2001, Biochemistry.

[19]  G. Patterson,et al.  Improved Fluorescence and Dual Color Detection with Enhanced Blue and Green Variants of the Green Fluorescent Protein* , 1998, The Journal of Biological Chemistry.

[20]  P. Schultz,et al.  Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Peter G Schultz,et al.  Unnatural amino acid mutagenesis of green fluorescent protein. , 2003, The Journal of organic chemistry.

[22]  S J Remington,et al.  Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein. , 1997, Biochemistry.

[23]  Robert Huber,et al.  Expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins. , 2003, Journal of molecular biology.

[24]  R. Tsien,et al.  Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer , 1996, Current Biology.

[25]  R. Heim,et al.  Understanding structure-function relationships in the Aequorea victoria green fluorescent protein. , 1999, Methods in cell biology.

[26]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[27]  M. Sisido,et al.  A non‐natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe , 2001, FEBS letters.

[28]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.