Rosalie L. M. Teeuwen

Single-Step Azide Introduction in Proteins via an Aqueous Diazo Transfer

The controlled introduction of azides in proteins provides targetable handles for selective protein manipulation. We present here an efficient diazo transfer protocol that can be applied in an aqueous solution, leading to the facile introduction of azides in the side chains of lysine residues and at the N-terminus of enzymes, e.g. horseradish peroxidase (HRP) and the red fluorescent protein DsRed. The effective introduction of azides was verified by mass spectrometry, after which the azido-proteins were used in Cu(I)-catalyzed [3 + 2] cycloaddition reactions. Azido-HRP retained its catalytic activity after conjugation of a small molecule. This modified protein could also be successfully immobilized on the surface of an acetylene-covered polymersome. Azido-DsRed was coupled to an acetylene-bearing protein allowing it to act as a fluorescent label, demonstrating the wide applicability of the diazo transfer procedure.

One-step photochemical attachment of NHS-terminated monolayers onto silicon surfaces and subsequent functionalization.

N-Hydroxysuccinimide (NHS)-ester-terminated monolayers were covalently attached in one step onto silicon using visible light. This mild photochemical attachment, starting from omega-NHS-functionalized 1-alkenes, yields a clean and flat monolayer-modified silicon surface and allows a mild and rapid functionalization of the surface by substitution of the NHS-ester moieties with amines at room temperature. Using a combination of analytical techniques (infrared reflection absorption spectroscopy (IRRAS), extensive X-ray photoelectron spectroscopy (XPS) in combination with density functional theory calculations of the XPS chemical shifts of the carbon atoms, atomic force microscopy (AFM), and static contact angle measurements), it was shown that the NHS-ester groups were attached fully intact onto the surface. The surface reactivity of the NHS-ester moieties toward amines was qualitatively and quantitatively evaluated via the reaction with para-trifluoromethyl benzylamine and biotin hydrazide.

“Clickable” elastins: elastin-like polypeptides functionalized with azide or alkyne groups

Elastin-like polypeptides (ELPs) functionalized with azide or alkyne groups were produced biosynthetically and coupled via the Cu-catalyzed azide-alkyne cycloaddition to a variety of (bio)molecules.

Elastin-like polypeptides of different molecular weights show independent transition temperatures when mixed

Elastin-like polypeptides (ELPs) with varying degrees of polymerization were produced viaprotein engineering. Lower critical solution temperatures of aqueous solutions containing two or three of these different molecular weight ELPs were investigated. In contrast to elastin-based side-chain polymers (EBPs) linear polypeptides preserve their individual transition temperature upon mixing.

Temperature-controlled positioning of fusion proteins in microreactors

We present a non-covalent immobilization system based on stimulus-responsive elastin-like polypeptides (ELPs) to facilitate the positioning of proteins in microchannels. Two ELP variants were constructed and connected to fluorescent proteins EGFP and DsRed2. These fusion proteins display an inverse transition behavior that can be simply controlled by varying concentrations of NaCl. With these ELP fusion proteins two patches of fluorescent proteins can be formed inside a microreactor, using only the temperature-responsive property of ELPs.