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The effect of charged versus neutral N- and C-termini on the stability of the collagen triple helix was examined. Thermal denaturation studies at different pH with collagen model peptides showed that an ammonium group at the N-terminus destabilizes the triple helix more than a carboxylate at the C-terminus. A neutral carboxylic acid stabilizes the triple helix more than an amido moiety at the C-terminus.

(4S)- and (4R)-configured aminoproline (Amp) residues were used as pH-responsive probes to tune the thermal stability of collagen triple helices in acidic and basic environments. The different steric and stereoelectronic properties of amino versus ammonium groups lead to a switch of the ring pucker of Amp upon changing the pH. The choice of the position of Amp within collagen model peptides (CMPs) as well as the absolute configuration at C(4) of the pH-responsive probe allows for tuning of the stability of Amp-containing collagen triple helices over a broad range. Comparative quantum chemical calculations on the steric and stereoelectronic effects of amino and ammonium groups versus fluorine, hydroxy, chlorine, and methyl substituents support the experimental findings. The research also shows that substitution of the naturally occurring hydroxy group in collagen by electron-withdrawing groups with a larger hydration shell than that of the hydroxy group is not tolerated.

(4S)-Aminoproline is introduced as a pH-sensitive probe for tuning the conformational properties of peptides and proteins. The pH-triggered flip of the ring puckering and the formation/release of a transannular H bond were used to switch the formation of collagen triple helices on and off reversibly.

We report a lipid-based strategy to visualize Golgi structure and dynamics at super-resolution in live cells. The method is based on two novel reagents: a trans-cyclooctene-containing ceramide lipid (Cer-TCO) and a highly reactive, tetrazine-tagged near-IR dye (SiR-Tz). These reagents assemble via an extremely rapid ‘tetrazine-click’ reaction into Cer-SiR, a highly photostable ‘vital dye’ that enables prolonged live cell imaging of the Golgi apparatus by 3D confocal and STED microscopy. Cer-SiR is non-toxic at concentrations as high as 2 μM and does not perturb the mobility of Golgi-resident enzymes or the traffic of cargo from the endoplasmic reticulum through the Golgi and to the plasma membrane.

Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution., The intracellular applications of STED microscopy are limited by the availability of dyes. Here the authors develop a two-colour labelling strategy based on SiR and ATTO590 dyes, and apply their strategy to image various subcellular membrane compartments.

NMR spectroscopic studies with a series of proline derivatives revealed that the polarity of the environment has a significant effect on the trans : cis isomer ratio of Xaa–Pro bonds. Computational studies showed that this effect is due to differences in the overall dipole moments of trans and cis conformers. Comparisons between the conformational properties of amide and ester derivatives revealed an intricate balance between polarity effects and n → π* interactions of adjacent carbonyl groups. The findings have important implications for protein folding and signaling as well as the performance of proline-based stereoselective catalysts., Chemical Science, 6 (12), ISSN:2041-6520, ISSN:2041-6539

The article summarizes our research devoted to the development of functionalizable collagen using azidoproline-containing model peptides. 'Click chemistry' or Staudinger reduction followed by acylation provided facile access to a range of differently functionalized collagen model peptides bearing e.g. carbohydrates. The research provided not only insight into the factors that are responsible for the high conformational stability of collagen but also a guide in which positions functional moieties are tolerated without or by deliberately disturbing the supramolecular assembly of collagen. The presented research will be useful for the development of collagen-based materials.

The effect of sterically demanding groups at proline residues on the conformational stability of the collagen triple helix was examined. The thermal stabilities (T(m) and ΔG) of eight different triple helices derived from collagen model peptides with (4R)- or (4S)-configured amidoprolines bearing either methyl or bulkier tert-butyl groups in the Xaa or Yaa position were determined and served as a relative measure for the conformational stability of the corresponding collagen triple helices. The results show that sterically demanding substituents are tolerated in the collagen triple helix when they are attached to (4R)-configured amidoprolines in the Xaa position or to (4S)-configured amidoprolines in the Yaa position. Structural studies in which the preferred conformation of (4R)- or (4S)-configured amidoproline were overlaid with the Pro and Hyp residues within a crystal structure of collagen revealed that the sterically demanding groups point to the outside of these two triple helices and thereby do not interfere with the formation of the triple helix. In all of the other examined collagen derivatives with lower stability of the triple helices, the acetyl or pivaloyl residues point toward the inside of the triple helix and clash with a residue of the neighboring strand. The results also revealed that unfavorable steric dispositions affect the conformational stability of the collagen triple helix more than unfavorable ring puckers of the proline residues. The results are useful for the design of functionalized collagen based materials.

The functionalizability and conformational properties of azidoproline (Azp)-containing collagen model peptides (CMPs) were studied. The results show that (4R)Azp has a similar stabilizing effect on the collagen triple helix as (4R)hydroxyproline and that functionalized CMPs are readily accessible by “click” chemistry. The resulting triazole-functionalized CMPs form stable triple helices, demonstrating that sterically demanding moieties in three symmetry-related positions in all strands are tolerated. The straightforward synthesis and facile functionalizability of the Azp-containing CMPs are intriguing for the development of functional collagen-based materials.

An 'azido gauche effect' determines the conformation of (4S)- and (4R)-azidoproline (Azp) derivatives and affects the s-cis:s-trans conformer ratio of Xaa-Azp bonds. The article summarizes our research on the conformational analysis of monomers as well as oligomers derived from (4S)Azp and (4R)Azp. We show that (4S)Azp and (4R)Azp can be used to tune the stability of the polyproline II (PPII) helix. In addition we demonstrate that Azpcontaining oligoprolines are attractive molecular scaffolds with a well-defined helical conformation that can be readily further functionalized using e.g. click chemistry.