Site-Specific Incorporation of Novel Backbone Structures into Proteins

Abstract

A number of unnatural amino acids and amino acid analogs with modified backbone structures were substituted for alanine-82 in T4 lysozyme. Replacements included alpha,alpha-disubstituted amino acids, N-alkyl amino acids, and lactic acid, an isoelectronic analog of alanine. The effects of these electronic and structural perturbations on the stability of T4 lysozyme were determined. The relatively broad substrate specificity of the Escherichia coli protein biosynthetic machinery suggests that a wide range of backbone and side-chain substitutions can be introduced, allowing a more precise definition of the factors affecting protein stability.

The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center

“…During continuous translation, however, P site-bound peptidyl-D-aa-tRNAs partition ECs into two subpopulations, a productive subpopulation that is competent for further rounds of translation elongation and a nonproductive subpopulation that is translationally arrested. This arrested subpopulation, which results in truncated polypeptide products containing a D-amino acid at their C termini due to the failure of the P site-bound peptidyl-D-aa-tRNA to act as a peptidyl-transferase donor, provides the most likely explanation for the poor unnatural amino acid incorporation efficiencies that have been observed during attempts to synthesize proteins containing D-Tyr (8), D-Trp (14), D-Arg (14), and possibly other unnatural amino acids (5).…”

“…Biochemical studies with these same aa-tRNAs demonstrated that D-Tyr-tRNA Tyr bound more weakly to elongation factor (EF) Tu than L-Tyr-tRNA Tyr and that D-Tyr was incorporated into the nascent polypeptide by the ribosome at a slower rate and with a lower yield than L-Tyr (13). More recent studies have used engineered suppressor tRNAs (i.e., in vitro transcribed tRNAs lacking all posttranscriptional modifications that read through stop or sense codons) and have reported D-amino acid incorporation efficiencies ranging from 0% to greater than 40% (4,5,14,15), depending on the in vitro translation system used (i.e., comprised of a cell extract or of fully purified components), the identities of the engineered tRNA and D-amino acid used, and the inclusion or exclusion of DTD.…”

“…Motivated by the observation that the TM can incorporate D-amino acids into proteins but does so with relatively low incorporation efficiencies (4,5,14,15), we sought to elucidate at which step(s) during the elongation cycle D-amino acids are discriminated against by the TM and determine the mechanism through which this discrimination occurs. To this end, we have prepared D-aa-tRNAs and evaluated their performance as substrates for the TM during discrete steps of the elongation cycle using a highly active in vitro translation system composed of purified translation components and lacking DTD (16).…”

“…Nonetheless, a comprehensive mechanistic understanding of how the translational machinery (TM) responds to D-aa-tRNAs is of interest for several reasons. First, improved incorporation of D-amino acids by the TM would be useful for protein engineering applications that seek to use the synthetic power of the ribosome to create novel polymers (4) as well as for mechanistic applications that seek to probe protein structure and folding with unnatural amino acids (5). Second, there is growing evidence that ribosomes may have to contend with D-aa-tRNAs in vivo: D-amino acids are synthesized by racemase enzymes (6) and can be found at high concentrations in cells (7), and a growing number of aa-tRNA synthetase (aaRS) enzymes exhibit the ability to misacylate tRNAs with D-amino acids (8,9).…”

Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor

“…Recently, a method was described that allowed the introduction of noncoded amino acids by introducing a stop codon. It used a modified tRNA that is acylated with the unnatural amino acid and has a recognition site complementary to the stop codon (Ellman et al, 1992). Another approach is the replacement of peptide fragments in a protein with the help of proteases (Chaiken, 1981).…”