Quartz crystal microbalance sensor for the detection of collagen model peptides based on the formation of triple helical structure.

Collagen is a major structural protein, and abnormalities in collagen structure can lead to several connective tissue diseases such as osteoporosis. We report the preparation of a collagen sensor using a synthetic peptide as proof of concept for detecting the collagen like peptides. The synthetic peptide 9-fluorenylmethyloxycarbonyl (Fmoc)-(prolyl-prolyl-glycine)7-OH was coupled to thiazolidine, which gets adsorbed on metal surfaces. Fmoc-(prolyl-prolyl-glycine)7-thiazolidine was immobilized on the surface of a quartz crystal microbalance (QCM) electrode used as a sensor probe. The collagen model peptide (prolyl-prolyl-glycine)10 could be detected, and the model peptide was directly adsorbed onto the surface of the electrode and was not removed by washing with hot water. Additionally, it was proved that the sensitivity of the probe could be enhanced to nanogram order by immobilizing the blocking reagent, Fmoc-prolyl-prolyl-glycine, within the gap of sensor probes on the electrode. The detectable mass of the model peptide decreased as the probe gap became narrower because of self-association of the probes. Moreover, the sensitivity of sensor probes also decreases as the gap between the probes becomes wider. Therefore, the optimum distance between the immobilized probes was determined from the simulation based on the experimental values. The association rate of the model peptide with sensor probes could be quantitatively determined when the distance between the probes was optimum, and this result suggested that most sensor probes could form a triple helical structure with the model peptide.

Conformational preferences of 4-chloroproline residues.

Conformational preferences of the (2S,4R)-4-chloroproline (Clp) and (2S,4S)-4-chloroproline (clp) residues are explored at the M06-2X/cc-pVTZ//M06-2X/6-31+G(d) level of theory in the gas phase and in water, where solvation free energies were calculated using the implicit solvation model, and by an X-ray diffraction study in the solid state. In the gas phase, the down-puckered γ-turn structure with the trans prolyl peptide bond is most preferred for both Ac-Clp-NHMe and Ac-clp-NHMe, in which the C(7) hydrogen bond between two terminal groups seems to play a role, as found for Ac-Pro-NHMe. In water, the Clp residue has a strong preference for the up-puckered PP(II) structure, whereas the up-puckered PP(II) structure prevails a little over the down-puckered PP(II) structure for the clp residue, similar to the Pro residue. Hence, our calculated results on the puckering preference of the Clp and clp residues in water are in accord with the observed results deduced from the relative stabilities of the triple helices of the collagen model peptides. The X-ray structure of Ac-clp-NHMe was found to be the most preferred in water but that of Ac-Clp-NHMe was located as a local minimum with ΔG = 2.0 kcal/mol. In particular, the X-ray structure of Ac-Clp-NHMe was quite different from that of Ac-Clp-OMe but similar to that of Ac-Pro-NHMe. The lowest rotational barriers to the prolyl cis-trans isomerization for Ac-Clp-NHMe become nearly the same as those for Ac-Pro-NHMe in water, whereas the barriers are lower by ∼2 kcal/mol for Ac-clp-NHMe. It was found that the cis-trans isomerization may proceed through the clockwise or anticlockwise rotations for Ac-Clp-NHMe and the anticlockwise rotation for Ac-clp-NHMe and Ac-Pro-NHMe in water.

Polymorphism of collagen triple helix revealed by 19F NMR of model peptide [Pro-4(R)-hydroxyprolyl-Gly]3-[Pro-4(R)-fluoroprolyl-Gly]-[Pro-4(R)-hydroxyprolyl-Gly]3.

We have characterized various structures of (Pro-Hyp(R)-Gly)(3)-Pro-fPro(R)-Gly-(Pro-Hyp(R)-Gly)(3) in the process of cis-trans isomerization and helix-coil transition by exploiting the sole (19)F NMR probe in 4(R)-fluoroproline (fPro(R)). Around the transition temperature (T(m)), we detected a species with a triple helical structure distinct from the ordinary one concerning the alignment of three strands. The (19)F-(19)F exchange spectroscopy showed that this misaligned and that the ordinary triple helices were interchangeable only indirectly via an extended monomer strand with all-trans peptide bonds at Pro-fPro(R), Pro-Hyp(R), and Gly-Pro in the central segment. This finding demonstrates that the helix-coil transition of collagen peptides is not described with a simple two-state model. We thus elaborated a scheme for the transition mechanism of (Pro-Hyp(R)-Gly)(n) that the most extended monomer strand can be the sole source both to the misaligned and correctly folded triple-helices. The staggered ends could help misaligned triple helices to self-assemble to higher-order structures. We have also discussed the possible relationship between the misaligned triple helix accumulating maximally at T(m) and the kinetic hysteresis associated with the helix-coil transition of collagen.

The triple helical structure and stability of collagen model peptide with 4(S)-hydroxyprolyl-Pro-Gly units.

Extensive studies on the structure of collagen have revealed that the hydroxylation of Pro residues in a variety of model peptides with the typical (X-Y-Gly)(n) repeats (X and Y: Pro and its analogues) represents one of the major factors influencing the stability of triple helices. While(2S,4R)-hydroxyproline (Hyp) at the position Y stabilizes the triple helix, (2S,4S)-hydroxyproline (hyp) at the X-position destabilizes the helix as demonstrated that the triple helix of (hyp-Pro-Gly)(15) is less stable than that of (Pro-Pro-Gly)(15) and that a shorter peptide (hyp-Pro-Gly)(10) does not form the helix. To clarify the role of the hydroxyl group of Pro residues to play in the stabilization mechanism of the collagen triple helix, we synthesized and crystallized a model peptide (Pro-Hyp-Gly)(4) -(hyp-Pro-Gly)(2) -(Pro-Hyp-Gly)(4) and analyzed its structure by X-ray crystallography and CD spectroscopy. In the crystal, the main-chain of this peptide forms a typical collagen like triple helix. The majority of hyp residues take down pucker with exceptionally shallow angles probably to relieve steric hindrance, but the remainders protrude the hydroxyl group toward solvent with the less favorable up pucker to fit in a triple helix. There is no indication of the existence of an intra-molecular hydrogen bond between the hydroxyl moiety and the carbonyl oxygen of hyp supposed to destabilize the triple helix. We also compared the conformational energies of up and down packers of the pyrrolidine ring in Ac-hyp-NMe(2) by quantum mechanical calculations.

Conformational analysis of human calcitonin in solution.

The solution conformation of human calcitonin in a mixture of 60% water and 40% trifluoroethanol has been determined by the combined use of 1H NMR spectroscopy and distance geometry calculations with a distributed computing technique. 1H NMR spectroscopy provided 195 distance constraints and 13 hydrogen bond constraints. The 20 best converged structures exhibit atomic rmsd of 0.43 A for the backbone atoms from the averaged coordinate position in the region of Asn3-Phe22. The conformation is characterized by a nearly amphiphilic alpha-helix domain that extends from Leu4 in the cyclic region to His20. There are no significant differences observed among the overall structures of a series of calcitonins obtained from ultimobranchial bodies, including those that possess 20- to 50-fold greater activity. Three aromatic amino acid residues, Tyr12, Phe16 and Phe19, form a hydrophobic surface of human calcitonin. Bulky side chains on the surface could interfere with the ligand-receptor interaction thereby causing its low activity, relative to those of other species.

Collagen-like triple helix formation of synthetic (Pro-Pro-Gly)10 analogues: (4(S)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10, (4(R)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10 and (4(S)-fluoroprolyl-4(R)-fluoroprolyl-Gly)10.

For the rational design of a stable collagen triple helix according to the conventional rule that the pyrrolidine puckerings of Pro, 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) should be down at the X-position and up at the Y-position in the X-Y-Gly repeated sequence for enhancing the triple helix propensities of collagen model peptides, a series of peptides were prepared in which X- and Y-positions were altogether occupied by Hyp(R), Hyp(S), fPro(R) or fPro(S). Contrary to our presumption that inducing the X-Y residues to adopt a down-up conformation would result in an increase in the thermal stability of peptides, the triple helices of (Hyp(S)-Hyp(R)-Gly)(10) and (fPro(S)-fPro(R)-Gly)(10) were less stable than those of (Pro-Hyp(R)-Gly)(10) and (Pro-fPro(R)-Gly)(10), respectively. As reported by Bächinger's and Zagari's groups, (Hyp(R)-Hyp(R)-Gly)(10) which could have an up-up conformation unfavorable for the triple helix, formed a triple helix that has a high thermal stability close to that of (Pro-Hyp(R)-Gly)(10). These results clearly show that the empirical rule based on the conformational preference of pyrrolidine ring at each of X and Y residues should not be regarded as still valid, at least for predicting the stability of collagen models in which both X and Y residues have electronegative groups at the 4-position.

Different effects of 4-hydroxyproline and 4-fluoroproline on the stability of collagen triple helix.

Differential scanning calorimetry (DSC) analyses of a series of collagen model peptides suggest that 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) have different effects on the stability of the collagen triple helices according to the sequence of amino acids and stereochemistry at the 4 positions of these imino acids. The thermodynamic parameters indicate that the enhanced stabilities are classified into two different types: the enthalpy term is primarily responsible for the enhanced stability of the triple helix of (Pro-Hyp(R)-Gly)(10), whereas the entropy term dominates the enhanced stability of (Pro-fPro(R)-Gly)(10). The difference between the molecular volumes observed in solution and intrinsic molecular volumes calculated from the crystal structure indicates the different hydration states of these peptides. (Pro-Hyp(R)-Gly)(10) is highly hydrated compared to (Pro-Pro-Gly)(10), which contributes to the larger enthalpy. In contrast, the volume of (Pro-fPro(R)-Gly)(10) shows a smaller degree of hydration than that of (Pro-Pro-Gly)(10). The entropic cost of forming the triple helix of the fPro-containing peptides is compensated by a decrease in an ordered structure of water molecules surrounding the peptide molecule, although the contribution of enthalpy originating from the hydration is reduced. These arguments about the different contribution of entropic and enthalpic terms were successfully applied to interpret the stability of the triple helix of (fPro(S)-Pro-Gly)(10) as well.

Characterization of collagen model peptides containing 4-fluoroproline; (4(S)-fluoroproline-pro-gly)10 forms a triple helix, but (4(R)-fluoroproline-pro-gly)10 does not.

Collagen model peptide (Pro-Pro-Gly)10 has a triple helical structure and undergoes a thermal transition to a single random coil structure. The transition temperature of the analogous model peptides depends largely on amino acid substitution. Substitution of Pro by 4-hydroxyproline (Hyp) or 4-fluoroproline (fPro) has especially attracted attention because the position of substitution and chirality of the hydroxyl group or fluorine atom affect the transition temperatures. Here, we demonstrated that (4(S)-fPro-Pro-Gly)10 takes a triple helical structure, but (4(R)-fPro-Pro-Gly)10 exists in a single chain structure. This is not consistent with the case of Hyp substitution in our previous report where both (4(S)-Hyp-Pro-Gly)10 and (4(R)-Hyp-Pro-Gly)10 are in a single random coil state.