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.

Linear conjuncted porphyrin trimer synthesis via "click" reaction

Two isomers of porphyrin trimer with 1,4-diaryltriazole linkers have been synthesized using "click" methodology and characterized by MS, NMR and UV-vis spectroscopy. The porphyrin compounds were shown to exhibit photostability and high solubility. Photophysical data were compared with corresponding ones of synthesized triazole-bridged dimer with the same electronic surrounding and known linear dimer and trimer with diarylethyne linkers. Obtained data revealed weak inter-chromophore electronic communication in the ground state but significant exciton coupling whereas the properties of the individual chromophores are mainly retained for triazole-bridged dimer and trimers.

Infrared Study of Acetic Acid Solutions in CCl4

Spectra of acetic acid solutions in carbon tetrachloride were taken at room temperature over the concentration range 0.025-0.00125 mol dm-3. Solutions of acetic acid were modelled as an ideal mixture of monomers, and cyclic and linear dimers. Regression analysis effected separation of the experimental envelope into its component bands. Band shapes were approximated by a Lorenzian function with the resolved band peak frequencies 1 712 cm-1 for the cyclic dimer, 1 724 cm-1 for the linear dimer and 1 765 cm-1 for the acetic acid monomer. Mean values of equilibrium constants for the standard state of unit concentration are 2 700 for the cyclic and 393 for the linear dimer, respectively.

Genetic and physical analysis of plasmid recombination in recB recC sbcB and recB recC sbcA Escherichia coli K-12 mutants.

The effect of mutations in known recombination genes (recA, recB, recC, recE, recF, recJ, recN, recO, recQ and ruv) on intramolecular recombination of plasmids was studied in recB recC sbcB and recB recC sbcA Escherichia coli mutants. The rate of recombination of circular dimer plasmids was at least 1000-fold higher in recB recC sbcB or recB recC sbcA mutants as compared to wild-type cells. The rate was decreased by mutations in recA, recF, recJ, recO, ruv or mutS in recB recC sbcB mutants, and by mutations in recE, recN, recO, recQ, ruv or mutS in recB recC sbcA mutants. In addition to measuring the recombination rate of circular dimer plasmids, the recombination-mediated transformation of linear dimer plasmids was also studied. Linear dimer plasmids transformed recB recC sbcB and recB recC sbcA mutants 20- to 40-fold more efficiently than wild-type cells. The transformation efficiency of linear dimer plasmids in recB recC sbcB mutants was decreased by mutations in recA, recF, recJ, recO, recQ or lexA (lexA3). In recB recC sbcA mutants the transformation efficiency of linear dimers was decreased only by a recE mutation. Physical analysis of linear dimer- or circular dimer-transformed recB recC sbcB mutants revealed that all transformants contained recombinant monomer genotypes. This suggests that recombination in recB recC sbcB cells is very efficient.

Diolefincobalt(0)-Komplexe / Diolefincobalt(0) Complexes

Mononuclear cobalt(0) complexes containing diolefin and trimethylphosphane ligands Co(cycloocta-1,5-diene)(PMe3)2 and Co[(CH2=CHMe2Si)2O](PMe3)2 are obtained from CoCl2 by reaction with magnesium in the presence of the ligands or by substitution of cobalt(olefin)tris- (trimethylphosphane) with diolefin. Chemical and spectroscopic properties confirm the para- magnetic valence state of the compounds in solution. An X-Ray crystal structure determination of Co[(CH2=CHMe2Si)2O](PMe3)2 shows a tetrahedral arrangement of ligand functions around the cobalt atom. With butadiene a diamagnetic dinuclear complex [Co(C4H6)(PMe3)3]2 is obtained containing π-allyl co-ordination of two Co(PMe3)3 units to a linear dimer of butadiene