Broadening the Utility of Farnesyltransferase-Catalyzed Protein Labeling Using Norbornene-Tetrazine Click Chemistry.

Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification, in which one or two 15- or 20-carbon isoprenoid chains are transferred onto cysteine residues near the C-terminus of a target protein. The three main enzymes─protein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II)─that catalyze this process have been shown to tolerate numerous structural modifications in the isoprenoid substrate. This feature has previously been exploited to transfer an array of farnesyl diphosphate analogues with a range of functionalities, including an alkyne-containing analogue for copper-catalyzed bioconjugation reactions. Reported here is the synthesis of an analogue of the isoprenoid substrate embedded with norbornene functionality (C10NorOPP) that can be used for an array of applications, ranging from metabolic labeling to selective protein modification. The probe was synthesized in seven steps with an overall yield of 7% and underwent an inverse electron demand Diels-Alder (IEDDA) reaction with tetrazine-containing tags, allowing for copper-free labeling of proteins. The use of C10NorOPP for the study of prenylation was explored in the metabolic labeling of prenylated proteins in HeLa, COS-7, and astrocyte cells. Furthermore, in HeLa cells, these modified prenylated proteins were identified and quantified using label-free quantification (LFQ) proteomics with 25 enriched prenylated proteins. Additionally, the unique chemistry of C10NorOPP was utilized for the construction of a multiprotein-polymer conjugate for the targeted labeling of cancer cells. That construct was prepared using a combination of norbornene-tetrazine conjugation and azide-alkyne cycloaddition, highlighting the utility of the additional degree of orthogonality for the facile assembly of new protein conjugates with novel structures and functions.

Bis(2-nitro-phen-yl)methane.

In the title compound, C13H10N2O4, the nitro groups are twisted significantly relative to the benzene rings [dihedral angles = 16.64 (18) and 28.02 (11)°]. The benzene groups are nearly perpendicular to each other [dihedral angle = 87.72 (6)°]. Short inter-molecular N⋯O and C⋯O [2.981 (2) and 3.060 (2) Å, respectively] contacts suggest possible weak π-inter-actions between nitro groups and between benzene and nitro groups. In addition, there are π-π inter-actions between one benzene group and an inversion-related equivalent [inter-planar separation = 3.494 (2) Å].

Site-specific labeling of proteins and peptides with trans-cyclooctene containing handles capable of tetrazine ligation.

There is a growing library of functionalized non-natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non-natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions. To date the most common strategy used in this approach has been copper catalyzed azide-alkyne cycloaddition (CuAAC). While being fast and bioorthogonal CuAAC has limited use in live cell experiments due to copper's toxicity.(1) Here, we report the synthesis of trans-cyclooctene geranyl diphosphate. This substrate can be synthesized from geraniol in six steps and be enzymatically transferred to peptides and proteins that end in a CAAX sequence. Proteins and peptides site-specially modified with trans-cyclooctene geranyl diphosphate were subsequently targeted for further modification via tetrazine ligation. As tetrazine ligation is bioorthogonal, fast, and is contingent on ring strain rather than the addition of a copper catalyst, this labeling strategy should prove useful for labeling proteins where the presence of copper may hinder solubility or biological reactivity.

Engineering protein farnesyltransferase for enzymatic protein labeling applications.

Creating covalent protein conjugates is an active area of research due to the wide range of uses for protein conjugates spanning everything from biological studies to protein therapeutics. Protein Farnesyltransferase (PFTase) has been used for the creation of site-specific protein conjugates, and a number of PFTase substrates have been developed to facilitate that work. PFTase is an effective catalyst for protein modification because it transfers Farnesyl diphosphate (FPP) analogues to protein substrates on a cysteine four residues from the C-terminus. While much work has been done to synthesize various FPP analogues, there are few reports investigating how mutations in PFTase alter the kinetics with these unnatural analogues. Herein we examined how different mutations within the PFTase active site alter the kinetics of the PFTase reaction with a series of large FPP analogues. We found that mutating either a single tryptophan or tyrosine residue to alanine results in greatly improved catalytic parameters, particularly in kcat. Mutation of tryptophan 102ß to alanine caused a 4-fold increase in kcat and a 10-fold decrease in KM for a benzaldehyde-containing FPP analogue resulting in an overall 40-fold increase in catalytic efficiency. Similarly, mutation of tyrosine 205ß to alanine caused a 25-fold increase in kcat and a 10-fold decrease in KM for a coumarin-containing analogue leading to a 300-fold increase in catalytic efficiency. Smaller but significant changes in catalytic parameters were also obtained for cyclo-octene- and NBD-containing FPP analogues. The latter compound was used to create a fluorescently labeled form of Ciliary Neurotrophic Factor (CNTF), a protein of therapeutic importance. Additionally, computational modeling was performed to study how the large non-natural isoprenoid analogues can fit into the active sites enlarged via mutagenesis. Overall, these results demonstrate that PFTase can be improved via mutagenesis in ways that will be useful for protein engineering and the creation of site-specific protein conjugates.

Investigation of the sequence and length dependence for cell-penetrating prenylated peptides.

Cell penetrating peptides are useful delivery tools for introducing molecules of interest into cells. A new class of cell penetrating molecules has been recently reported-cell penetrating, prenylated peptides. In this study a series of such peptides was synthesized to examine the relationship between peptide sequence and level of peptide internalization and to probe their mechanism of internalization. This study revealed that prenylated peptides internalize via a non-endocytotic pathway regardless of sequence. Sequence length and identity was found to play a role in peptide uptake but prenylated sequences as short as two amino acids were found to exhibit significant cell penetrating properties.

A minimalist substrate for enzymatic peptide and protein conjugation.

Recently a number of nonnatural prenyl groups containing alkynes and azides have been developed as handles to perform click chemistry on proteins and peptides ending in the sequence "CAAX", where C is a cysteine that becomes alkylated, A is an aliphatic amino acid and X is any amino acid. When such molecules are modified, a tag containing a prenyl analogue and the "CAAX box" sequence remains. Here we report the synthesis of an alkyne-containing substrate comprised of only nine nonhydrogen atoms. This substrate was synthesized in six steps from 3-methylbut-2-en-1-ol and has been enzymatically incorporated into both proteins and peptides by using protein farnesyltransferase. After prenylation the final three amino acids required for enzymatic recognition can be removed by using carboxypeptidase Y, leaving a single residue (the cysteine from the "CAAX box") and the prenyl analogue as the only modifications. We also demonstrate that this small tag minimizes the impact of the modification on the solubility of the targeted protein. Hence, this new approach should be useful for applications in which the presence of a large tag hinders the modified protein's solubility, reactivity, or utility.

Multifunctional prenylated peptides for live cell analysis.

Protein prenylation is a common post-translational modification present in eukaryotic cells. Many key proteins involved in signal transduction pathways are prenylated, and inhibition of prenylation can be useful as a therapeutic intervention. While significant progress has been made in understanding protein prenylation in vitro, we have been interested in studying this process in living cells, including the question of where prenylated molecules localize. Here, we describe the synthesis and live cell analysis of a series of fluorescently labeled multifunctional peptides, based on the C-terminus of the naturally prenylated protein CDC42. A synthetic route was developed that features a key Acm to Scm protecting group conversion. This strategy was compatible with acid-sensitive isoprenoid moieties and allowed incorporation of an appropriate fluorophore as well as a cell-penetrating sequence (penetratin). These peptides are able to enter cells through different mechanisms, depending on the presence or absence of the penetratin vehicle and the nature of the prenyl group attached. Interestingly, prenylated peptides lacking penetratin are able to enter cells freely through an energy-independent process and localize in a perinuclear fashion. This effect extends to a prenylated peptide that includes a full "CAAX box" sequence (specifically, CVLL). Hence, these peptides open the door for studies of protein prenylation in living cells, including enzymatic processing and intracellular peptide trafficking. Moreover, the synthetic strategy developed here should be useful for the assembly of other types of peptides that contain acid-sensitive functionalities.

Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose.

Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their nonprenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography medium that has been chemically functionalized with beta-cyclodextrin (beta-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target ("CAAX box") sequences were enzymatically prenylated in vitro with natural and nonnatural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a beta-CD-Sepharose column. One particular prenylation reaction, farnesylation of an mCherry-CAAX fusion construct, was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a nonnatural, functional isoprenoid substrate, the functional group was maintained by chromatography on beta-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, beta-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.