Aminotryptophan-containing barstar: Structure–function tradeoff in protein design and engineering with an expanded genetic code

2006 ◽  
Vol 1764 (7) ◽  
pp. 1147-1158 ◽  
Author(s):  
Marina Rubini ◽  
Sandra Lepthien ◽  
Ralph Golbik ◽  
Nediljko Budisa
Keyword(s):  
Structure Function ◽  
Genetic Code ◽  
Protein Design ◽  
Expanded Genetic Code ◽  
Protein Design And Engineering

scholarly journals Measuring the tolerance of the genetic code to altered codon size

2021 ◽  
Author(s):  
E. DeBenedictis ◽  
D. Söll ◽  
K. Esvelt
Keyword(s):  
Genetic Code ◽  
Protein Translation ◽  
Single Amino Acid ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases ◽  
Triplet Codon ◽  
Orthogonal Set ◽  
Expanded Genetic Code ◽  
Primordial Earth

SummaryProtein translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet-codon, rather than quadruplet-codon, genetic code? Here, we investigate the tolerance of the E. coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all twenty canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs), many of which selectively incorporate a single amino acid in response to a specified four-base codon. However, efficient quadruplet codon translation often requires multiple tRNA mutations, potentially constraining evolution. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results illuminate factors that led to selection and maintenance of triplet codons in primordial Earth and provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.

scholarly journals Making Sense of “Nonsense” and More: Challenges and Opportunities in the Genetic Code Expansion, in the World of tRNA Modifications

2022 ◽  
Vol 23 (2) ◽  
pp. 938
Author(s):  
Olubodun Michael Lateef ◽  
Michael Olawale Akintubosun ◽  
Olamide Tosin Olaoba ◽  
Sunday Ocholi Samson ◽  
Malgorzata Adamczyk
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Protein Design ◽  
Side Chains ◽  
Vast Number ◽  
Trna Modifications ◽  
Wide Range ◽  
Challenges And Opportunities ◽  
Code Extension ◽  
Genetic Code Expansion

The evolutional development of the RNA translation process that leads to protein synthesis based on naturally occurring amino acids has its continuation via synthetic biology, the so-called rational bioengineering. Genetic code expansion (GCE) explores beyond the natural translational processes to further enhance the structural properties and augment the functionality of a wide range of proteins. Prokaryotic and eukaryotic ribosomal machinery have been proven to accept engineered tRNAs from orthogonal organisms to efficiently incorporate noncanonical amino acids (ncAAs) with rationally designed side chains. These side chains can be reactive or functional groups, which can be extensively utilized in biochemical, biophysical, and cellular studies. Genetic code extension offers the contingency of introducing more than one ncAA into protein through frameshift suppression, multi-site-specific incorporation of ncAAs, thereby increasing the vast number of possible applications. However, different mediating factors reduce the yield and efficiency of ncAA incorporation into synthetic proteins. In this review, we comment on the recent advancements in genetic code expansion to signify the relevance of systems biology in improving ncAA incorporation efficiency. We discuss the emerging impact of tRNA modifications and metabolism in protein design. We also provide examples of the latest successful accomplishments in synthetic protein therapeutics and show how codon expansion has been employed in various scientific and biotechnological applications.

scholarly journals Protein evolution with an expanded genetic code

2008 ◽  
Vol 105 (46) ◽  
pp. 17688-17693 ◽  
Author(s):  
Chang C. Liu ◽  
Antha V. Mack ◽  
Meng-Lin Tsao ◽  
Jeremy H. Mills ◽  
Hyun Soo Lee ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Directed Evolution ◽  
Unnatural Amino Acids ◽  
Selective Advantage ◽  
Display System ◽  
Antibody Library ◽  
Evolution Of Proteins ◽  
Expanded Genetic Code ◽  
Selection Of

We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in VH CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded “synthetic” genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.

scholarly journals Exploring the potential impact of an expanded genetic code on protein function

2015 ◽  
Vol 112 (22) ◽  
pp. 6961-6966 ◽  
Author(s):  
Han Xiao ◽  
Fariborz Nasertorabi ◽  
Sei-hyun Choi ◽  
Gye Won Han ◽  
Sean A. Reed ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Conformational Changes ◽  
Protein Function ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
Functional Changes ◽  
Living Organisms ◽  
Expanded Genetic Code

With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing kcat, but not significantly affecting KM. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216–AcrF mutation leads to conformational changes in key active site residues—both in the free enzyme and upon formation of the acyl-enzyme intermediate—that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.

scholarly journals Bacteriophages use an expanded genetic code on evolutionary paths to higher fitness

2014 ◽  
Vol 10 (3) ◽  
pp. 178-180 ◽  
Author(s):  
Michael J Hammerling ◽  
Jared W Ellefson ◽  
Daniel R Boutz ◽  
Edward M Marcotte ◽  
Andrew D Ellington ◽  
...  
Keyword(s):  
Genetic Code ◽  
Evolutionary Paths ◽  
Expanded Genetic Code
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