Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions.

Enzymes are of central importance to many biotechnological and biomedical applications. However, for many potential applications, the required conditions impede enzyme folding and therefore function. The enzyme Sortase A is a transpeptidase that is widely used to perform bioconjugation reactions with peptides and proteins. Thermal and chemical stress impairs Sortase A activity and prevents its application under harsh conditions, thereby limiting the scope for bioconjugation reactions. Here, we report the stabilization of a previously reported, activity-enhanced Sortase A, which suffered from particularly low thermal stability, using the in situ cyclization of proteins (INCYPRO) approach. After introduction of three spatially aligned solvent-exposed cysteines, a triselectrophilic cross-linker was attached. The resulting bicyclic INCYPRO Sortase A demonstrated activity both at elevated temperature and in the presence of chemical denaturants, conditions under which both wild-type Sortase A and the activity-enhanced version are inactive.

In Situ Cyclization of Proteins (INCYPRO): Cross-Link Derivatization Modulates Protein Stability.

Protein macrocyclization represents a very efficient strategy to increase the stability of protein tertiary structures. Here, we describe a panel of novel C3-symmetric tris-electrophilic agents and their use for the cyclization of proteins. These electrophiles are reacted with a protein domain harboring three solvent-exposed cysteine residues, resulting in the in situ cyclization of the protein (INCYPRO). We observe a clear dependency of cross-linking rates on the electrophilicity. All nine obtained cross-linked protein versions show considerably increased thermal stability (up to 29 °C increased melting temperature) when compared to that of the linear precursor. Most interestingly, the degree of stabilization correlates with the hydrophilicity of the cross-link. These results will support the development of novel cross-linked proteins and enable a more rational design process.

Development of Ubiquitin-Based Probe for Metalloprotease Deubiquitinases.

Deubiquitinases (DUBs) are a family of enzymes that regulate the ubiquitin signaling cascade by removing ubiquitin from specific proteins in response to distinct signals. DUBs that belong to the metalloprotease family (metalloDUBs) contain Zn2+ in their active sites and are an integral part of distinct cellular protein complexes. Little is known about these enzymes because of the lack of specific probes. Described here is a Ub-based probe that contains a ubiquitin moiety modified at its C-terminus with a Zn2+ chelating group based on 8-mercaptoquinoline, and a modification at the N-terminus with either a fluorescent tag or a pull-down tag. The probe is validated using Rpn11, a metalloDUB found in the 26S proteasome complex. This probe binds to metalloDUBs and efficiently pulled down overexpressed metalloDUBs from a HeLa cell lysate. Such probes may be used to study the mechanism of metalloDUBs in detail and allow better understanding of their biochemical processes.

Reactive-site-centric chemoproteomics identifies a distinct class of deubiquitinase enzymes.

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.

Development of a hypersensitive periodate-cleavable amino acid that is methionine- and disulfide-compatible and its application in MHC exchange reagents for T cell characterisation.

Incorporation of cleavable linkers into peptides and proteins is of particular value in the study of biological processes. Here we describe the synthesis of a cleavable linker that is hypersensitive to oxidative cleavage as the result of the periodate reactivity of a vicinal amino alcohol moiety. Two strategies directed towards the synthesis of a building block suitable for solid-phase peptide synthesis were developed: a chemoenzymatic route, involving L-threonine aldolase, and an enantioselective chemical route; these led to α,γ-diamino-ß-hydroxybutanoic acids in diastereoisomerically mixed and enantiopure forms, respectively. Incorporation of the 1,2-amino alcohol linker into the backbone of a peptide generated a conditional peptide that was rapidly cleaved at very low concentrations of sodium periodate. This cleavable peptide ligand was applied in the generation of MHC exchange reagents for the detection of antigen-specific T cells in peripheral blood cells. The extremely low concentration of periodate required to trigger MHC peptide exchange allowed the co-oxidation of methionine and disulfide residues to be avoided. Conditional MHC reagents hypersensitive to periodate can now be applied without limitations when UV irradiation is undesired or less practical.

Carbosilane dendrimeric carbodiimides: site isolation as a lactamization tool.

The convergent syntheses of three generations of carbosilane dendrimeric carbodiimides are described. The wedge-type building blocks were synthesized in a divergent way, starting from allyl chloride and a repetitive sequence of hydrosilylation with HSiCl3 and a Grignard reaction with allylmagnesium bromide. Hydrogenation of the terminal double bonds led to inert and stable wedges. The chloride substituent at the focal point was transformed into several functional groups that eventually led to dendrimeric structures with a carbodiimide core. The extent of the site isolation effect of the dendrimers was studied with dilution experiments monitored by FT-IR spectroscopy on the corresponding dendrimeric ureas. These studies showed that only the first generation self-aggregates via hydrogen bonding, while the second and the third do not, implying isolation of core-bound moieties. The dendrimeric carbodiimides mediated lactamization reactions to obtain homodiketopiperazines.