Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

Natural backbone-cyclized proteins have an increased thermostability and resistance towards proteases, characteristics that have sparked interest in head-to-tail cyclization as a method to stability-enhance proteins used in diagnostics and therapeutic applications, for example. In this proof-of principle study, we have produced and investigated a head-to-tail cyclized and HER2-specific ZHER2:342 Affibody dimer. The sortase A-mediated cyclization reaction is highly efficient (>95%) under optimized conditions, and renders a cyclic ZHER3:342-dimer with an apparent melting temperature, Tm, of 68 °C, which is 3 °C higher than that of its linear counterpart. Circular dichroism spectra of the linear and cyclic dimers looked very similar in the far-UV range, both before and after thermal unfolding to 90 °C, which suggests that cyclization does not negatively impact the helicity or folding of the cyclic protein. The cyclic dimer had an apparent sub-nanomolar affinity (Kd ~750 pM) to the HER2-receptor, which is a ~150-fold reduction in affinity relative to the linear dimer (Kd ~5 pM), but the anti-HER2 Affibody dimer remained a high-affinity binder even after cyclization. No apparent difference in proteolytic stability was detected in an endopeptidase degradation assay for the cyclic and linear dimers. In contrast, in an exopeptidase degradation assay, the linear dimer was shown to be completely degraded after 5 min, while the cyclic dimer showed no detectable degradation even after 60 min. We further demonstrate that a site-specifically DyLight 594-labeled cyclic dimer shows specific binding to HER2-overexpressing cells. Taken together, the results presented here demonstrate that head-to-tail cyclization can be an effective strategy to increase the stability of an Affibody dimer.

Features of polymerization of methylmethacrylate using a photocatalyst – the complex oxide RbTe1.5W0.5O6

Radical polymerization of methylmethacrylate in an aqueous emulsion was carried out using the complex oxide RbTe 1.5 W 0.5 O 6 as a photoinitiator under visible light irradiation with λ= 400-700 nm. Studies of the process and reaction products using modern methods of physical and chemical analysis (GPC, IR, NMR, etc.) have shown that several directions of monomer transformations occur simultaneously in the reaction mixture. Polymethylmethacrylate, produced in the organic phase and characterized by Mn ~ 140-145 kDa, is a result of polymerization initiation by a hydroxyl radical formed due to complex transformations of electron-hole pairs during irradiation of the photocatalyst. Moreover, the interaction of the hydroxyl radical with OH groups on the complex oxide RbTe 1.5 W 0.5 O 6 surface and the subsequent formation of oxygen-centered radicals lead to grafting polymer macromolecules onto the photocatalyst surface. In addition, methylmethacrylate is able to oxidize to a cyclic dimer with terminal double bonds and then form a polymer with cyclic dimer links due to coordination by double bonds on the complex oxide RbTe 1.5 W 0.5 O 6 surface. The high activity of the hydroxyl radical made it possible to obtain the graft copolymer PMMA-pectin by grafting the polymer product onto the surface of the natural polymer-pectin. Comparison of the sponge morphology of the graft copolymer PMMA-pectin and the initial pectin samples using the scanning electron microscopy method showed a noticeable difference in their structural and topological organization. This fact is especially interesting in terms of studying the properties of the graft copolymer as a material for the scaffolds.

Aggregation-induced Chirality Amplification of Optically Active Fluorescent Polyurethane and Cyclic Dimer in the Ground and Excited States

Optically active linear polyurethane and cyclic dimer were synthesized from 2,7-diisocyanatofluorene and 2,2’-dihydroxy-1,1’-binaphthyl. Circular dichroism (CD) spectral intensity of polymer was amplified at a higher concentration through aggregate formation while...

Azetidin-2-ones: structures of antimitotic compounds based on the 1-(3,4,5-trimethoxyphenyl)azetidin-2-one core

A series of related substituted 1-(3,4,5-trimethoxyphenyl)azetidin-2-ones have been characterized: 3-(4-fluorophenyl)-4-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one, C25H24FNO5 (1), 3-(furan-2-yl)-4-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one, C23H23NO6 (2), 4-(4-methoxyphenyl)-3-(naphthalen-1-yl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one, C29H27NO5 (3), 3-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one, C27H29NO7 (4) and 4,4-bis(4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-one, C32H31NO6 (5). All of the compounds are racemic. The lactam and 3,4,5-trimethoxyphenyl rings are approximately co-planar and the orientation of the lactam and the 4-methoxyphenyl substituent is approximately orthogonal. The chiral centres, although eclipsed by geometry, have torsion angles ranging from −7.27 to 13.08° for the 3 position, and −8.69 to 13.76° for the 4 position of the β-lactam. The structures display intramolecular C—H...O bonding between the 3,4,5-trimethoxyphenyl ring and the lactam ketone. Further C—H...O interactions are observed and form either an opposing methoxy `buckle' to join two molecules together or a cyclic dimer.

Reversible hydrogen-bonded polymerization regulated by allosteric metal templation

A stable quadruple hydrogen bonded cyclic dimer assisted by metal templation was successfully self-assembled and its reversible transformation to supramolecular polymer was investigated.

Relationship between solid state structure and solution stability of copper(ii)–hydroxypyridinecarboxylate complexes

More acidic –OH groups increase the solution stability of copper(ii)–hydroxypyridinecarboxylate complexes and they lose the bridging role in cyclic dimer formation.