Towards controlling activity of a peptide asparaginyl ligase (PAL) by lumazine synthetase compartmentalization.

Peptide asparaginyl ligases (PALs) hold significant potential in protein bioconjugation due to their excellent kinetic properties and broad substrate compatibility. However, realizing their full potential in biocatalytic applications requires precise control of their activity. Inspired by nature, we aimed to compartmentalize a representative PAL, OaAEP1-C247A, within protein containers to create artificial organelles with substrate sorting capability.

Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion.

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice.

Deciphering the Synthetic and Refolding Strategy of a Cysteine-Rich Domain in the Tumor Necrosis Factor Receptor (TNF-R) for Racemic Crystallography Analysis and d-Peptide Ligand Discovery.

Many cell-surface receptors are promising targets for chemical synthesis because of their critical roles in disease development. This synthetic approach enables investigations by racemic protein crystallography and ligand discovery by mirror-image methodologies. However, due to their complex nature, the chemical synthesis of a receptor can be a significant challenge.

Preparing recombinant "Split AEP" for protein labeling.

A variant originated from Oldenlandia affinis asparaginyl ligase, OaAEP1-C247A, has emerged as an ideal tool for protein labeling. However, its preparation was laborious and time-consuming. It is recombinantly produced as a zymogen, requiring acid activation and four chromatographic steps; despite these extensive steps, the catalytically active enzyme exhibited only moderate purity.

Investigating the effects of cyclic topology on the performance of a plastic degrading enzyme for polyethylene terephthalate degradation.

Agitation is a commonly encountered stress for enzymes during all stages of production and application, but investigations that aim to improve their tolerance using topological engineering have yet to be reported. Here, the plastic-degrading enzyme IsPETase was cyclized in a range of topologies including a cyclic monomer, cyclic dimer and catenane using SpyTag/SpyCatcher technologies, and their tolerance towards different stresses including mechanical agitation was investigated. The cyclic dimer and catenane topologies were less susceptible to agitation-induced inactivation resulting in enhancement of polyethylene terephthalate (PET) degradation.

Towards the use of an amino acid cleavable linker for solid-phase chemical synthesis of peptides and proteins.

The synthesis of proteins by solid-phase chemical ligation (SPCL) suffers from the paucity of linkers that can be cleaved under mild conditions. Here, we deployed a spontaneous nickel-assisted cleavage (SNAC) tag, known to undergo spontaneous cleavage in the presence of nickel(II), as a linker for C-to-N SPCL.

D-Peptide and D-Protein Technology: Recent Advances, Challenges, and Opportunities.

Total chemical protein synthesis provides access to entire D-protein enantiomers enabling unique applications in molecular biology, structural biology, and bioactive compound discovery. Key enzymes involved in the central dogma of molecular biology have been prepared in their D-enantiomeric forms facilitating the development of mirror-image life. Crystallization of a racemic mixture of L- and D-protein enantiomers provides access to high-resolution X-ray structures of polypeptides.

Computational design of an amidase by combining the best electrostatic features of two promiscuous hydrolases.

While there has been emerging interest in designing new enzymes to solve practical challenges, computer-based options to redesign catalytically active proteins are rather limited. Here, a rational QM/MM molecular dynamics strategy based on combining the best electrostatic properties of enzymes with activity in a common reaction is presented. The computational protocol has been applied to the re-design of the protein scaffold of an existing promiscuous esterase from Bs2 to enhance its secondary amidase activity.

Effect of Trimethine Cyanine Dye- and Folate-Conjugation on the In Vitro Biological Activity of Proapoptotic Peptides.

Despite continuous advances, anticancer therapy still faces several technical hurdles, such as selectivity on cellular and subcellular targets of therapeutics. Toward addressing these limitations, we have combined the use of proapoptotic peptides, trimethine cyanine dye, and folate to target the mitochondria of tumor cells. A series of proapoptotic peptides and their conjugates with a cyanine dye and/or folate were synthesized in the solid phase, and their toxicity in different human cell lines was assessed.

Spatio-Temporal Photoactivation of Cytotoxic Proteins.

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light.

The role of streptavidin and its variants in catalysis by biotinylated secondary amines.

Here, we combine the use of host screening, protein crystallography and QM/MM molecular dynamics simulations to investigate how the protein structure affects iminium catalysis by biotinylated secondary amines in a model 1,4 conjugate addition reaction. Monomeric streptavidin (M-Sav) lacks a quaternary structure and the solvent-exposed reaction site resulted in poor product conversion in the model reaction with low enantio- and regioselectivities. These parameters were much improved when the tetrameric host T-Sav was used; indeed, residues at the symmetrical subunit interface were proven to be critical for catalysis through a mutagenesis study.

Asparaginyl endopeptidases: enzymology, applications and limitations.

Asparaginyl endopeptidases (AEP) are cysteine proteases found in mammalian and plant cells. Several AEP isoforms from plant species were found to exhibit transpeptidase activity which is integral for the key head-to-tail cyclisation reaction during the biosynthesis of cyclotides. Since many plant AEPs exhibit excellent enzyme kinetics for peptide ligation via a relatively short substrate recognition sequence, they have become appealing tools for peptide and protein modification.

Approaches for peptide and protein cyclisation.

The cyclisation of polypeptides can play a crucial role in exerting biological functions, maintaining stability under harsh conditions and conferring proteolytic resistance, as demonstrated both in nature and in the laboratory. To date, various approaches have been reported for polypeptide cyclisation. These approaches range from the direct linkage of N- and C- termini to the connection of amino acid side chains, which can be applied both in reaction vessels and in living systems.

Cryo-kinetics Reveal Dynamic Effects on the Chemistry of Human Dihydrofolate Reductase.

Effects of isotopic substitution on the rate constants of human dihydrofolate reductase (HsDHFR), an important target for anti-cancer drugs, have not previously been characterized due to its complex fast kinetics. Here, we report the results of cryo-measurements of the kinetics of the HsDHFR catalyzed reaction and the effects of protein motion on catalysis. Isotopic enzyme labeling revealed an enzyme KIE (k /k ) close to unity above 0 °C; however, the enzyme KIE was increased to 1.

Transfer hydrogenations catalyzed by streptavidin-hosted secondary amine organocatalysts.

Here, the streptavidin-biotin technology was applied to enable organocatalytic transfer hydrogenation. By introducing a biotin-tethered pyrrolidine (1) to the tetrameric streptavidin (T-Sav), the resulting hybrid catalyst was able to mediate hydride transfer from dihydro-benzylnicotinamide (BNAH) to α,β-unsaturated aldehydes. Hydrogenation of cinnamaldehyde and some of its aryl-substituted analogues was found to be nearly quantitative.

Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency.

Genetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair.

Enabling protein-hosted organocatalytic transformations.

In this review, the development of organocatalytic artificial enzymes will be discussed. This area of protein engineering research has underlying importance, as it enhances the biocompatibility of organocatalysis for applications in chemical and synthetic biology research whilst expanding the catalytic repertoire of enzymes. The approaches towards the preparation of organocatalytic artificial enzymes, techniques used to improve their performance (selectivity and reactivity) as well as examples of their applications are presented.

Electric Field Measurements Reveal the Pivotal Role of Cofactor-Substrate Interaction in Dihydrofolate Reductase Catalysis.

The contribution of ligand-ligand electrostatic interaction to transition state formation during enzyme catalysis has remained unexplored, even though electrostatic forces are known to play a major role in protein functions and have been investigated by the vibrational Stark effect (VSE). To monitor electrostatic changes along important steps during catalysis, we used a nitrile probe (T46C-CN) inserted proximal to the reaction center of three dihydrofolate reductases (DHFRs) with different biophysical properties, DHFR (EcDHFR), its conformationally impaired variant (EcDHFR-S148P), and DHFR (BsDHFR). Our combined experimental and computational approach revealed that the electric field projected by the substrate toward the probe negates those exerted by the cofactor when both are bound within the enzymes.

Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling.

Asparaginyl endopeptidases (AEPs) are ideal for peptide and protein labeling. However, because of the reaction reversibility, a large excess of labels or backbone modified substrates are needed. In turn, simple and cheap reagents can be used to label N-terminal cysteine, but its availability inherently limits the potential applications.

Exploring the Chemoselectivity towards Cysteine Arylation by Cyclometallated Au Compounds: New Mechanistic Insights.

To gain more insight into the factors controlling efficient cysteine arylation by cyclometallated Au complexes, the reaction between selected gold compounds and different peptides was investigated by high-resolution liquid chromatography electrospray ionization mass spectrometry (HR-LC-ESI-MS). The deduced mechanisms of C-S cross-coupling, also supported by density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) calculations, evidenced the key role of secondary peptidic gold binding sites in favouring the process of reductive elimination.

Streptavidin-Hosted Organocatalytic Aldol Addition.

In this report, the streptavidin-biotin technology was applied to enable organocatalytic aldol addition. By attaching pyrrolidine to the valeric motif of biotin and introducing it to streptavidin (Sav), a protein-based organocatalytic system was created, and the aldol addition of acetone with -nitrobenzaldehyde was tested. The conversion of substrate to product can be as high as 93%.