Birgit Wiltschi

Synthetic Biology of Proteins: Tuning GFPs Folding and Stability with Fluoroproline

Background Proline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the Cγ-exo or -endo puckering of their pyrrolidine rings. Peptide bond conformation as well as puckering propensity can be manipulated by proper choice of ring substituents, e.g. Cγ-fluorination. Synthetic chemistry has routinely exploited ring-substituted proline analogs in order to change, modulate or control folding and stability of peptides. Methodology/Principal Findings In order to transmit this synthetic strategy to complex proteins, the ten proline residues of enhanced green fluorescent protein (EGFP) were globally replaced by (4R)- and (4S)-fluoroprolines (FPro). By this approach, we expected to affect the cis/trans peptidyl-proline bond isomerization and pyrrolidine ring puckering, which are responsible for the slow folding of this protein. Expression of both protein variants occurred at levels comparable to the parent protein, but the (4R)-FPro-EGFP resulted in irreversibly unfolded inclusion bodies, whereas the (4S)-FPro-EGFP led to a soluble fluorescent protein. Upon thermal denaturation, refolding of this variant occurs at significantly higher rates than the parent EGFP. Comparative inspection of the X-ray structures of EGFP and (4S)-FPro-EGFP allowed to correlate the significantly improved refolding with the Cγ-endo puckering of the pyrrolidine rings, which is favored by 4S-fluorination, and to lesser extents with the cis/trans isomerization of the prolines. Conclusions/Significance We discovered that the folding rates and stability of GFP are affected to a lesser extent by cis/trans isomerization of the proline bonds than by the puckering of pyrrolidine rings. In the Cγ-endo conformation the fluorine atoms are positioned in the structural context of the GFP such that a network of favorable local interactions is established. From these results the combined use of synthetic amino acids along with detailed structural knowledge and existing protein engineering methods can be envisioned as a promising strategy for the design of complex tailor-made proteins and even cellular structures of superior properties compared to the native forms.

Inhibition of the fungal fatty acid synthase type I multienzyme complex

Fatty acids are among the major building blocks of living cells, making lipid biosynthesis a potent target for compounds with antibiotic or antineoplastic properties. We present the crystal structure of the 2.6-MDa Saccharomyces cerevisiae fatty acid synthase (FAS) multienzyme in complex with the antibiotic cerulenin, representing, to our knowledge, the first structure of an inhibited fatty acid megasynthase. Cerulenin attacks the FAS ketoacyl synthase (KS) domain, forming a covalent bond to the active site cysteine C1305. The inhibitor binding causes two significant conformational changes of the enzyme. First, phenylalanine F1646, shielding the active site, flips and allows access to the nucleophilic cysteine. Second, methionine M1251, placed in the center of the acyl-binding tunnel, rotates and unlocks the inner part of the fatty acid binding cavity. The importance of the rotational movement of the gatekeeping M1251 side chain is reflected by the cerulenin resistance and the changed product spectrum reported for S. cerevisiae strains mutated in the adjacent glycine G1250. Platensimycin and thiolactomycin are two other potent inhibitors of KSs. However, in contrast to cerulenin, they show selectivity toward the prokaryotic FAS system. Because the flipped F1646 characterizes the catalytic state accessible for platensimycin and thiolactomycin binding, we superimposed structures of inhibited bacterial enzymes onto the S. cerevisiae FAS model. Although almost all side chains involved in inhibitor binding are conserved in the FAS multienzyme, a different conformation of the loop K1413–K1423 of the KS domain might explain the observed low antifungal properties of platensimycin and thiolactomycin.

Lipase Congeners Designed by Genetic Code Engineering

Classical enzyme optimization exploits the chemistry confined to the 20 canonical amino acids encoded by the standard genetic code. ‘Genetic code engineering’ allows the global substitution of particular residues with synthetic analogues, endowing proteins with chemical diversity not found in nature. These proteins are congeners of the parent protein because they originate from the same gene sequence, but contain a fraction of noncanonical amino acids. Global substitutions of methionine, proline, phenylalanine, and tyrosine have been carried out with related analogues in Thermoanaerobacter thermohydrosulfuricus lipase. This study represents the first extensive report of an important biocatalyst substituted with a high number of noncanonical amino acids. The generated lipase congeners displayed special features such as enhanced activation, elevated enzyme activity (by up to 25 %) and substrate tolerance (by up to 40 %), and changes in optimal temperature (by up to 20 °C) and pH (by up to 3). These emergent features achieved by genetic code engineering might be important not only for academic research, but also for numerous economical applications in the food, detergent, chemical, pharmaceutical, leather, textile, cosmetic, and paper industries.

Performance Analysis of Orthogonal Pairs Designed for an Expanded Eukaryotic Genetic Code

Background The suppression of amber stop codons with non-canonical amino acids (ncAAs) is used for the site-specific introduction of many unusual functions into proteins. Specific orthogonal aminoacyl-tRNA synthetase (o-aaRS)/amber suppressor tRNACUA pairs (o-pairs) for the incorporation of ncAAs in S. cerevisiae were previously selected from an E. coli tyrosyl-tRNA synthetase/tRNACUA mutant library. Incorporation fidelity relies on the specificity of the o-aaRSs for their ncAAs and the ability to effectively discriminate against their natural substrate Tyr or any other canonical amino acid. Methodology/Principal Findings We used o-pairs previously developed for ncAAs carrying reactive alkyne-, azido-, or photocrosslinker side chains to suppress an amber mutant of human superoxide dismutase 1 in S. cerevisiae. We found worse incorporation efficiencies of the alkyne- and the photocrosslinker ncAAs than reported earlier. In our hands, amber suppression with the ncAA containing the azido group did not occur at all. In addition to the incorporation experiments in S. cerevisiae, we analyzed the catalytic properties of the o-aaRSs in vitro. Surprisingly, all o-aaRSs showed much higher preference for their natural substrate Tyr than for any of the tested ncAAs. While it is unclear why efficiently recognized Tyr is not inserted at amber codons, we speculate that metabolically inert ncAAs accumulate in the cell, and for this reason they are incorporated despite being weak substrates for the o-aaRSs. Conclusions/Significance O-pairs have been developed for a whole plethora of ncAAs. However, a systematic and detailed analysis of their catalytic properties is still missing. Our study provides a comprehensive scrutiny of o-pairs developed for the site-specific incorporation of reactive ncAAs in S. cerevisiae. It suggests that future development of o-pairs as efficient biotechnological tools will greatly benefit from sound characterization in vivo and in vitro in parallel to monitoring intracellular ncAA levels.

Residue-specific global fluorination of Candida antarctica lipase B in Pichia pastoris

We report the in vivo fluorination of the tryptophan, tyrosine, and phenylalanine residues in a glycosylation-deficient mutant of Candida antarctica lipase B, CalB N74D, expressed in the methylotrophic yeast Pichia pastoris and subsequently segregated into the growth medium. To achieve this, a P. pastoris strain auxotrophic for all three aromatic amino acids was supplemented with 5-fluoro-L-tryptophan, meta-fluoro-(DL)-tyrosine, or para-fluoro-L-phenylalanine during expression of CalB N74D. The residue-specific replacement of the canonical amino acids by their fluorinated analogs was confirmed by mass analysis. Although global fluorination induced moderate changes in the secondary structure of CalB N74D, the fluorous variant proteins were still active lipases. However, their catalytic activity was lower than that of the non-fluorinated parent protein while their resistance to proteolytic degradation by proteinase K remained unchanged. Importantly, we observed that the global fluorination prolonged the shelf life of the lipase activity, which is an especially useful feature for the storage of, e.g., therapeutic proteins. Our study represents the first step on the road to the production of biotechnologically and pharmacologically relevant fluorous proteins in P. pastoris.

Non-canonical amino acids as a useful synthetic biological tool for lipase-catalysed reactions in hostile environments

The incorporation of several non-canonical amino acids into the Thermoanaerobacter thermohydrosulfuricus lipase confers not only activity enhancement upon treatment with organic solvents (by up to 450%) and surfactants (resp. 1630%), but also protective effects against protein reducing (resp. 140%), alkylating (resp. 160%), and denaturing (resp.190%) agents as well as inhibitors (resp. 40%). This approach offers novel chemically diversified biocatalysts for hostile environments.

In vivo engineering of proteins with nitrogen-containing tryptophan analogs

Recently, it has become possible to reprogram the protein synthesis machinery such that numerous noncanonical amino acids can be translated into target sequences yielding tailor-made proteins. The canonical amino acid tryptophan (Trp) encoded by a single nucleotide triplet (UGG) is a particularly interesting target for protein engineering and design. Trp-residues can be substituted with a variety of analogs and surrogates generated biosynthetically or by organic chemistry. Among them, nitrogen-containing tryptophan analogs occupy a central position, as they have distinct chemical properties in comparison with aliphatic amines and imines. They resemble purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. These special properties of the analogs can be directly transmitted into related protein structures via in vivo ribosome-mediated translation. Proteins expressed in this way are further endowed with unique properties like new spectral, altered redox and titration features or might serve as useful biomaterials. We present and discuss current works and future developments in protein engineering with nitrogen-containing tryptophan analogs and related compounds as well as their relevance for academic and applicative research.

Expanding the genetic code of Saccharomyces cerevisiae with methionine analogues

We replaced the single N‐terminal methionine in heterologously expressed human Cu/Zn superoxide dismutase with the non‐canonical methionine analogues homopropargylglycine and norleucine in the yeast Saccharomyces cerevisiae. Our non‐canonical amino acid incorporation protocol involves a two‐step procedure. In the first step, the methionine auxotrophic yeast cells are accumulated in synthetic medium containing methionine while the target protein production is shut off. After a short methionine depletion phase, the cells are transferred to inducing medium that contains the methionine analogue instead of methionine and target protein expression is switched on. The initially low level incorporation of ∼12% could be elevated to 40% by increasing the non‐canonical amino acid concentration in the medium by 10‐fold. With this approach we were able to produce up to 5 mg substituted protein per litre of yeast culture. Copyright

Fine tuning the N-terminal residue excision with methionine analogues.

Protein biosynthesis starts with methionine (Met) in all living cells. In the cytosol of eukaryotes and archaebacteria, protein translation is initiated with Met whereas N-formylmethionine (fMet) is used in eubacteria, the mitochondria and chloroplasts. The formyl group is added to the free a-amino group of Met-tRNA by the methionyl-tRNA formyltransferase (EC 2.1.2.9) and fMet-tRNA participates in translation. Met is always the first amino acid incorporated into the N-terminal position of a protein, even if alternative start codons, such as GUG and UUG are used for translation initiation. However, the amino acid sequence of mature proteins seldom contains Met at the first position; the N termini of most proteins are modified by a variety of co-, and post-translational processing events. Peptide deformylase (EC 3.5.1.88) removes the N-formyl group from proteins in prokaryotes and organelles. The unmasked amino terminal Met can subsequently be cleaved from prokaryotic as well as eukaryotic proteins by the Met aminopeptidase (MetAP; EC 3.4.11.18). Additionally, novel N termini are generated by the enzymatic cleavage of leader sequences, or by proteolytic digest at one or more positions within the polypeptide chain. The covalent attachment of diverse chemical functionalities, such as acetyl-, phosphate-, and myristoylgroups, or ubiquitin represents other N-terminal modifications beyond proteolytic processing. 13] As most N-terminal Met residues are coor post-translationally removed, their function lies in translation initiation rather than in structure. Depending on the organism, between 55 and 70 % of the proteins are subject to N-terminal Met excision (NME) by MetAP. NME is an irreversible cotranslational process that occurs as soon as the first N-terminal residues of the nascent peptide protrude from the ribosomal exit tunnel, before protein folding starts to occur. 15, 16] In eukaryotic cells, this enzymatic processing coincides with the intracellular localization of MetAP close to the ribosome. Although the localization of bacterial MetAP is still unclear (C. Klein, personal communication), it is reasonable to suppose that it is ribosome associated as well. MetAP specifically cleaves Met in the first position of the precursor protein. Meinnel and coworkers showed that model peptides with N-terminal Leu and Phe could be processed by Escherichia coli MetAP (EcMAP1) in vitro but with considerably lower catalytic efficiency than Met. The cleavage efficiency of Met in the first position largely depends on the bulkiness and nature of the side chain of the second amino acid (Figure 1). In vivo and in vitro data analyses provided convincing evidence that NME is likely to occur if the second residue of the precursor is Ala, Cys, Gly, Pro or Ser. Met in the first position can also be excised if Thr or Val follows in position 2, but with lower efficiency. Excision of the N-terminal Met with Ile and Asn at the second position is inefficient but may occur if the sequence context (vide infra) supports it. NME is not observed with the other amino acids at the second position. 13, 18] Although the data were collected predominantly in in vitro studies with EcMAP1 or in vivo with proteins from E. coli, MetAPs from diverse organisms all have the same specificity. 18] Taken together, smaller amino acids, especially Ala, at the second position optimally support N-terminal Met excision, whereas large side chains at the second position block the action of MetAP (Figure 1). According to earlier studies, the third position in the precursor protein does not influence NME. However, recently it was shown that not only the amino acid in position 2 but additional residues following the first Met have a major impact on NME efficiency. In vitro excision of the first Met from model peptides with either Ala or Gly in the second position was optimal if Trp, Met, or Ser followed in the third position. In conFigure 1. N-terminal Met excision (NME) is an irreversible cotranslational modification that is completed before the nascent polypeptide chain is fully synthesized. The basic rule is that small amino acid residues in the second position after the N-terminal Met facilitate NME, whereas bulky residues inhibit the process. MetAP, Met aminopeptidase.

Binding of Small Mono- and Oligomeric Integrin Ligands to Membrane-Embedded Integrins Monitored by Surface Plasmon-Enhanced Fluorescence Spectroscopy

We recently developed a binding assay format by incorporating native transmembrane receptors into artificial phospholipid bilayers on biosensor devices for surface plasmon resonance spectroscopy. By extending the method to surface plasmon-enhanced fluorescence spectroscopy (SPFS), sensitive recording of the association of even very small ligands is enabled. Herewith, we monitored binding of synthetic mono- and oligomeric RGD-based peptides and peptidomimetics to integrins alphavbeta3 and alphavbeta5, after having confirmed correct orientation and functionality of membrane-embedded integrins. We evaluated integrin binding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimer revealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic was also found to be highly active with a slightly higher selectivity toward alphavbeta3. The different compounds were also evaluated in in vitro cell adhesion tests for their capacity to interfere with alphavbeta3-mediated cell attachment to vitronectin. We hereby demonstrated that the different RGD monomers were similarly effective; the RGD dimer and tetramer showed comparable IC50 values, which were, however, significantly higher than those of the monomers. Best cell detachment from vitronectin was achieved by the peptidomimetic. The novel SPFS-binding assay platform proves to be a suitable, reliable, and sensitive method to monitor the binding capacity of small ligands to native transmembrane receptors, here demonstrated for integrins.