Hydrophobic Moieties Bestow Fast-Folding and Hyperstability on Collagen Triple Helices.

Trans amide bonds and fast cis- trans isomerization of Xaa-Pro bonds are crucial for the stability and folding rate of collagen, the most abundant protein in mammals. Here, we explored the effect of pendant hydrophobic moieties on the folding and stability of collagen triple helices. Kinetic studies with a series of collagen model peptides showed that a local hydrophobic environment accelerates cis- trans isomerization to an extent that thermally induced unfolding and folding of the collagen triple helix take place at the same speed. Thermal denaturation studies revealed that the hydrophobic appendages provide hyperstable collagen triple helices ( Tm = 70 °C).

pH-Responsive Aminoproline-Containing Collagen Triple Helices.

(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.

Crystal structure of (4S)-aminoproline: conformational insight into a pH-responsive proline derivative.

The crystal structure of a (4S)-configured ammoniumproline derivative is presented. The trifluoroacetic acid salt of the acetylated methylester of (4S)-aminoproline (Ac-(4S)Amp-OMe) crystallized as the trans conformer with a C(4)-endo ring pucker. The high-resolution structure shows typical parameters for a transannular H-bond and an n → π* interaction between the adjacent amide and ester groups. The structure corroborates previous findings of solution phase studies on the importance of these two interactions in acidic and neutral aqueous environments for the conformation of this pH-responsive proline derivative.

Importance of dipole moments and ambient polarity for the conformation of Xaa-Pro moieties - a combined experimental and theoretical study.

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.

Switchable proline derivatives: tuning the conformational stability of the collagen triple helix by pH changes.

(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.

From azidoproline to functionalizable collagen.

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.