Synthetic enzymes for synthetic substrates

In recent years, hydrolases like cutinases, esterases and lipases have been recognized as powerful tools for hydrolysis of synthetic polymers such as polyethylene terephthalate (PET) as an environmentally friendly alternative for environmentally harmful chemical recycling methods1. PET is currently the most common type of aromatic polyester, with widespread application as packaging material, beverage bottles, and synthetic textile fibers. So far, cutinases have been the most active enzyme class regarding PET degradation. In nature, cutinases catalyze the hydrolysis of the aliphatic biopolyester cutin, the structural component of plant cuticle. Although cutinases are able to act on natural insoluble polyesters, their activities on non-natural substrates are quit low. For this reason, different engineering strategies were established to optimize “polyesterases” for synthetic polymers (Fig.1). Thereby, development of rationale enzyme-engineering strategies led to remarkable enhancement of hydrolytic activities on polyesters and clearly showed that the affinity between the enzyme and the substrate plays a key role in the enzymatic hydrolysis of synthetic polyester. Please click Additional Files below to see the full abstract

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.DFG, EXC 314, Unifying Concepts in Catalysi

Evaluation of bicinchoninic acid as a ligand for copper(I)-catalyzed azide-alkyne bioconjugations

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.The Cu(I)-catalyzed cycloaddition of terminal azides and alkynes (click chemistry) represents a highly specific reaction for the functionalization of biomolecules with chemical moieties such as dyes or polymer matrices. In this study we evaluate the use of bicinchoninic acid (BCA) as a ligand for Cu(I) under physiological reaction conditions. We demonstrate that the BCA–Cu(I)-complex represents an efficient catalyst for the conjugation of fluorophores or biotin to alkyne- or azide-functionalized proteins resulting in increased or at least equal reaction yields compared to commonly used catalysts like Cu(I) in complex with TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) or BPAA (bathophenanthroline disulfonic acid). The stabilization of Cu(I) with BCA represents a new strategy for achieving highly efficient bioconjugation reactions under physiological conditions in many application fields.EC/FP7/259043/EU/Computing Biomaterials/COMPBIOMATDFG, EXC 294, BIOSS Zentrum für Biologische Signalstudien - von der Analyse zur SyntheseDFG, GSC 4, Spemann Graduiertenschule für Biologie und Medizin (SGBM

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

Proline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the C(gamma)-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. C(gamma)-fluorination. Synthetic chemistry has routinely exploited ring-substituted proline analogs in order to change, modulate or control folding and stability of peptides.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 C(gamma)-endo puckering of the pyrrolidine rings, which is favored by 4S-fluorination, and to lesser extents with the cis/trans isomerization of the prolines.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 C(gamma)-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

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 tRNA CUA 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 detaile

Activation of Tyr, AzF, and AmF by TyrRS and the AzRSs.

<p>Tyr, AzF and AmF were used at a concentration of 5 mM. No amino acid was added to the negative control (w/o aa). TyrRS (A) was added at 1 µM and AzRS1 (B), AzRS3 (C), and AzRS6 (D) at a concentration of 5 µM. The data for each o-aaRS were collected in one series of experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#s4" target="_blank">Materials and Methods</a> for details). The average of duplicate determinations is shown; the bars indicate the discrete values.</p

Activation of Tyr, Bpa, AzF, and AmF by BpaRS.

<p>Tyr, Bpa, AzF and AmF were used at a concentration of 5 mM. In the negative control, the amino acid was omitted (w/o aa). BpaRS was added at a concentration of 3 µM. The data were all recorded in one row of experiments and each value was determined in duplicate. The bars denote the discrete values.</p

Structures of tyrosine and its analogs used in this study.

<p>Structures, names and abbreviations are shown.</p

ESI-MS analyses of selected hSOD1 variants.

<p>Only variant proteins for which defined mass spectra were obtained are shown. The same hSOD1 variants were detected on the immunoblot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone-0031992-g002" target="_blank">Figure 2</a>. The corresponding ESI-MS spectra are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone-0031992-g003" target="_blank">Figure 3</a>. All hSOD1 variants were found with the N-terminal methionine cleaved off and acetylated alanine at position 2, as reported in the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone.0031992-Hallewell1" target="_blank">[41]</a>. The occasionally attached sodium ions (+22.99 Da) most probably originated from the <i>Strep</i>-Tactin elution buffer which contained 150 mM NaCl. The buffer was not exchanged during sample concentration in order to avoid protein loss. In some of the protein preparations we found a known disulfide bond (S-S, −2 Da; between C57 and C146 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone.0031992-Lindberg1" target="_blank">[70]</a>).</p>1<p>All hSOD1 masses were calculated without N-terminal methionine, acetylated alanine at position 2 and with completely reduced cysteines.</p

Integrin-functionalized artificial membranes as test platforms for monitoring small integrin ligand binding by surface plasmon-enhanced fluorescence spectroscopy

The design and synthesis of molecularly or supramolecularly defined interfacial architectures have seen in recent years a remarkable growth of interest and scientific research activities for various reasons. On the one hand, it is generally believed that the construction of an interactive interface between the living world of cells, tissue, or whole organisms and the (inorganic or organic) materials world of technical devices such as implants or medical parts requires proper construction and structural (and functional) control of this organism–machine interface. It is still the very beginning of generating a better understanding of what is needed to make an organism tolerate implants, to guarantee bidirectional communication between microelectronic devices and living tissue, or to simply construct interactive biocompatibility of surfaces in general. This exhaustive book lucidly describes the design, synthesis, assembly and characterization, and bio-(medical) applications of interfacial layers on solid substrates with molecularly or supramolecularly controlled architectures. Experts in the field share their contributions that have been developed in recent years