Playing with the Molecules of Life

Abstract

Our understanding of the complex molecular processes of living organisms at the molecular level is growing exponentially. This knowledge, together with a powerful arsenal of tools for manipulating the structures of macromolecules, is allowing chemists to to harness and reprogram the cellular machinery in ways previously unimaged. Here we review one example in which the genetic code itself has been expanded with new building blocks that allow us to probe and manipulate the structures and functions of proteins with unprecedented precision.

Figure 1.

Expansion of the genetic code. A) Requisite components for the site-specific incorporation of ncAAs into proteins. Red coloring indicates primary sites of mutation required to enable the efficient incorporation of the desired ncAA. B) Site-specific incorporation of ncAAs through engineering of the translational machinery. Standard translation occurs with endogenous tRNA/aaRS pairs and canonical amino acids (blue) on the ribosome (yellow). An orthogonal tRNA/aaRS pair and ncAA are added (red) and encoded by a nonsense or frameshift codon on the mRNA (purple). Adapted from Kim, C.H. et. al 2013.

Figure 2.

Standard protocol for generation of an aaRS to encode ncAAs.

Figure 3.

Generation of an orthogonal ribosome. A) A non-orthogonal ribosome allows for cross talk between the two mRNAs, not providing efficient incorporation of ncAAs. B) An orthogonal ribosome where the endogenous system (grey) and the engineered ribosome and mRNA (green) exhibit no cross-reactivity. C) Crystal structure of the rRNA (orange), mRNA (purple) and tRNA (yellow), illustrating the key 530 loop within the ribosome that was subjected to mutagenesis to afford an orthogonal ribosome.

Figure 4.

Modulation of pKa and redox potential of tyrosine residues. A) The ribonucleotide reductase reaction converting ribose to deoxyribose relies upon a catalytic cysteine radical. The generation of this radical is dependent on radical formation on several key tyrosine residues. Altering the pKas and redox potentials of these residues affords key insights into the catalytic mechanism. B) Examples of ncAAs conferring altered tyrosine pKas and reduction potentials (E_p) that have been employed in the study of ribonucleotide reductase.

Figure 5.

Applications of photocrosslinking ncAAs. A) Structures of two common photocrosslinking ncAAs, p-benzoylphenylalanine and (3-(3-methyl-3H-diazirine-3-yl)-propaminocarbonyl-N^ε-lysine. B) Structure of the lipopolysaccharide transport protein E (LptE) with the sites of ncAA incorporation circled in red. C) Example of a photocrosslinking gel employing the benzophenone ncAA at multiple residues of the LptE protein associated with lipopolysaccharide transport. Gel shifts observed in the irradiated (+) samples indicate a crosslinking event with LptD. Proposed model of the LptD/LptE association for lipopolysaccharide transport established by the crosslinking experiments. Adapted from Freinkman, E. et al. 2011.

Figure 6.

Genetically encoding fluorescent amino acids. A) Structures of fluorescent ncAAs based on common fluorophores. B) Solvent sensitivity of ANAP fluorescence. C) Use of ANAP in a glutamine binding protein as a site-specific probe to detect glutamine binding by fluorescence. D) Demonstration of the use of an ANAP to track protein localization; the ncAA was incorporated into histones resulting in fluorescence only in nuclei. E) A FRET pair prepared via site-specific incorporation of ANAP (blue circle) in a fusion construct with GFP. The presence of ANAP obviates the need for a second fluorescent fusion protein.

Figure 7.

Using ncAAs as environmental sensors for biologically relevant analytes. A) Structures of ncAAs commonly employed in environmental sensors, including metal binders, H₂0₂, H₂S, and ONOO⁻ sensitive functionalities. B) General method for the development of an “on” sensor. Incorporation of the ncAA within the GFP chromophore alters or quenches fluorescence, however upon coordination or reaction with a desired analyte, the group is removed or fluorescence is shifted.

Figure 8.

Bioorthogonal conjugation with ncAAs. A) Common structures of ncAAs used in bioconjugations: p-azidophenylalanine (pAzF), p-propargyloxyphenylalanine (pPrF), and p-acetylphenylalanine (pAcF). B) General scheme of a bioorthogonal conjugation. The protein harboring an ncAA with unique reactivity is subjected to appropriate reaction conditions with another molecule (protein, DNA/RNA, surface, small molecule, etc; blue sphere) that possesses a chemical functionality that will only react with the ncAA and no other biological molecules. C) Incorporation of p-acetylphenylalanine into a Fab for Her2 followed by oxime ligation with DNA. This bioconjugate facilitates immuno-PCR with significantly higher levels of sensitivity than nonspecifically labeled antibodies. D) By site-specifically incorporating p-azidophenylalalanine GFP can be site-specifically immobilized on a solid support, conferring a higher degree of protein stability in non-aqueous solvents.

Figure 9.

Live cell imaging using ncAAs. A) Common structures of genetically encoded ncAAs for non-cytotoxic and rapid live cell imaging. Using either standard 1,3-cycloadditions with strained alkynes or Diels-Alder type reactions with a tetrazine and a strained alkyne or alkene, reactions can be performed within living cells. B) Genetic incorporation of the strained alkyne into vimentin for super-resolution, live cell imaging of proteins.

Figure 10.

Photocontrol over protein function with ncAAs. A) Photocaging strategy for activation of protein function with light. Typically a key residue is substituted with a caged ncAA, rendering the protein of interest non-functional. Upon brief irradiation with non-cytotoxic light, the caging group is removed to afford wild-type protein. X = O,N,S; R = H, O, OMe B) Photocaged tyrosine, lysine and serine amino acids with variations of the common o-nitrobenzyl caging group. C) Photocaging of the CRISPR/Cas9 system results in inactivity of the Cas9 protein, until brief irradiation with light restores its function close to WT levels, facilitating the expression of a reporter GFP plasmid. D) Photoswitchable regulation of protein function via reaction of a specifically placed ncAA with a ligand harboring an azobenzene based linker. Light irradiation of different wavelengths results in cis/trans isomerization, blocking or exposing the active site. E) Genetically-encoded decaging of a strained cycloalkene via reaction with a tetrazine, restoring a lysine residue and protein function. Proof-of-concept experiments were performed via caging luciferase, quenching luminescence until the tetrazine reagent is added.

Figure 11.

Therapeutic applications of ncAAs. A) Incorporation of p-acetylphenylalanine into an anti-CD3 Fab followed by oxime ligation with an aminooxy-modified DUPA produces a bispecific agent for the treatment of prostate cancer. Treatment of mice in tumor xenografts resulted in complete tumor killing. B) Example of using ncAAs to break immunological tolerance. The ncAA, p-nitrophenylalanine was incorporated into mTNF-α at distinct sites and used to immunize mice, resulting in increased survival relative to both the WT protein and a control in a endotoxemia mouse model.

Figure 12.

Computational design of ncAAs containing proteins. A) Structure of p-phenylphenylalanine (BipA) used in the model study B) Space filling model of the interactions of the biphenyl (green) locked into its planar transition state with the designed pocket.