Conformational change of the triple-helical structure. II. Conformation of (Pro-Pro-Gly)n and (Pro-Pro-Gly)n (Ala-Pro-Gly)m(Pro-Pro-Pro-Gly)n in an aqueous solution

Measurements of the molecular weight of (Pro-Pro-Gly)_n and (Pro-Pro-Gly)_n(Ala-Pro-Gly)_m(Pro-Pro-Gly)_n, which were synthesized by the solid-phase method, revealed that they formed a trimer in an aqueous solution, and dissociated into single-stranded chains on warming. Accompanying the transition, a large decrease of optical rotation was observed, like the collagen–gelatin transition. The shape of the trimeric molecule was rodlike, and the dimensions were 12 Å in diameter and 2.8 Å per residue in length, regardless of the length of Ala-Pro-Gly sequences in a peptide chain. The data indicate that both Pro-Pro-Gly sequences and Ala-Pro-Gly sequences from the triple-helical structure similar to that of collagen in aqueous solution. All optical rotational dispersion (ORD) curves of solutions of the peptides were represented by a single-term Drude equation, and the Drude constant λ_c was 200 nm for all peptides regardless of the length of Ala-Pro-Gly sequences. The resemblance between the helical structure formed by Pro-Pro-Gly sequences and that by Ala-Pro-Gly sequences was also suggested by the formation of the hybrid triple helix from two kinds of peptide chains with different lengths of Ala-Pro-Gly sequences.     

The formation of the triple helix in a mixed solution of (Pro-Pro-Gly)n and (Pro-Pro-Gly)n'. A model of molecular size recognition

A theoretical analysis is given of the triple-helix–random-coil transition in a mixed solution of poly(Pro-Pro-Gly)_n with two different but defined degrees of polymerization n and n′. Because of the highly cooperative nature of this helix–coil transition, each polypeptide chain tends to form a triple helix with other polypeptide chains with the same degree of polymerization (size recognition). Occurrence of triple helices consisting of polypeptide chains with different degrees of polymerization (error in recognition) is studied in detail as a function of the cooperativity, and n and n′. Implication of this analysis for molecular recognition in general is discussed.     

Two states of the triple helix in the thermal transition of the collagen model peptide (Pro-Pro-Gly)10

The collagen model peptide (Pro-Pro-Gly)10 is known to fold into a triple helix in solution. So far, the triple helix has been considered to exist as a single state. However, our previous study of (Pro-Pro-Gly)10 in solution has indicated the presence of two different states of the triple helix, a lower (HL) and a higher temperature state (HH). In the present study, these triple-helical states were investigated in more detail by NMR. Complete stereospecific assignments of the methylene protons of the proline residues were accomplished by the use of NOESY and TOCSY spectra. The temperature dependence of the 1H chemical shifts showed that the HL-to-HH thermal transition can be attributed to a conformational change of the first proline (Pro1) residues of the (Pro-Pro-Gly) triplets. Since TOCSY spectra with a 10 ms mixing-time confirmed a down puckering of these Pro residues in the HL state, but interconverting down and up puckerings in the HH state, the HL-to-HH thermal transition corresponds to conformational changes of the pyrrolidine rings of the Pro1 residues from an uniform down puckering to a more flexible state. The results confirm that thermal unfolding of the triple helix proceeds through the intermediate HH state.     

Vibrational neutron spectroscopy of collagen and model polypeptides.

A pulsed source neutron spectrometer has been used to measure vibrational spectra (20-4000 cm-1) of dry and hydrated type I collagen fibers, and of two model polypeptides, polyproline II and (prolyl-prolyl-glycine)10, at temperatures of 30 and 120 K. the collagen spectra provide the first high resolution neutron views of the proton-dominated modes of a protein over a wide energy range from the low frequency phonon region to the rich spectrum of localized high frequency modes. Several bands show a level of fine structure approaching that of optical data. The principal features of the spectra are assigned. A difference spectrum is obtained for protein associated water, which displays an acoustic peak similar to pure ice and a librational band shifted to lower frequency by the influence of the protein. Hydrogen-weighted densities of states are extracted for collagen and the model polypeptides, and compared with published calculations. Proton mean-square displacements are calculated from Debye-Waller factors measured in parallel quasi-elastic neutron-scattering experiments. Combined with the collagen density of states function, these yield an effective mass of 14.5 a.m.u. for the low frequency harmonic oscillators, indicating that the extended atom approximation, which simplifies analyses of low frequency protein dynamics, is appropriate.     

Synthesis and conformational study of sequential polypeptides, (L-ala-L-val-gly)n and (L-val-L-ala-gly)n.

As an approach for elucidating the role of sequences of amino acids in protein structures, model polypeptides having the same composition but different sequences of amino acids, (L -Ala-L -Val-Gly)_n and (L -Val-L -Ala-Gly)_n, have been prepared by the method involving facile monomer synthesis using N-carboxy α-amino acid anhydrides and N-hydroxysuccinimide esters. The yields and the molecular weights of the polypeptides formed by polycondensation do not depend on the monomer concentrations, but on the sequences of the amino acids in the monomers. Infrared spectra in the solid state showed that (L -Ala-L -Val-Gly)_n can take the α-helical conformation but (L -Val-L -Ala-Gly)_n cannot. The results suggest that the conformations of polypeptides are influenced by the sequences of the amino acids in the polypeptides.     

Comprehensive conformational analysis of (Gly-Pro-Pro)n and (Gly-Pro-Hyp)n related to collagen

A complete analysis of all possible conformations with correct hydrogen bonds of the collagen II type was performed on the basis of developed simultaneous equations. Using a unimodal search (by varying Ψ³), the energetically favorable structure was obtained. No other energetically satisfactory structural solutions are possible. The next aim was to obtain a precise model of the molecule. The program used includes a subroutine for continual deformation of the pyrrolidine rings. The set of parameters determining the structure consists of 14 independent variables (8 dihedral and 6 bond angles). As starting points for the energy optimization, conformations produced by scanning and some structures from previous work were used. The final structures (practically the same for both polymers) have helix parameters h = 0.285 nm and t = 52°, which are in excellent agreement with the 7/2 symmetry of diffraction data. The conformations of the pyrrolidine rings are of the B type, i.e., C₂-C^β-exo-C^γ-endo. For both polypeptides, the conformations of imino acids in position 3 of the triplet are the same; in position 2, however, they are slightly different. The difference in diffraction patterns for the 7/2 and 10/3 helices is discussed.     

Physicochemical analysis of (Pro-Pro-Gly)n with defined molecular weight--temperature dependence of molecular weight in aqueous solution

Sequenced polytripeptides, (Pro-Pro-Gly)_n, (n = 10, 15, 20), with defined molecular weights were synthesized by the solid-phase method. Conformational changes of these sample as a function of temperature were studied by measurements of optical rotation and sedimentation equilibrium. The temperature dependence of optical rotation was shown similar to thermal transition of collagen molecule. Each of these polymers existed as a timer at lower temperature. (Pro-Pro-Gly)₁₀ existed as a monomer at higher temperature, and the others were expected to behave analogously.     

Conformational change of the triple-helical structure. IV. Kinetics of the helix-folding of (Pro-Pro-Gly)n (n = 10, 12, and 15)

The kinetic curves of the helix-refolding of (PPG)_n (n = 10, 12, and 15) were analyzed with an all-or-none model. The Arrhenius plot of the overall rate constant of the helixfolding k_F showed a negative activation energy at high temperature. With the aid of a sequential model, it was concluded that the reason for the anomaly was the instability of short helices (shorter than seven helical units in a trimeric molecule), and/or the more rapid rates of helix-folding and helix-opening for shorter helices. The rate constant of the formation of one helical unit composed of three tripeptides at an end of a long helix was calculated to be 10^2–4 sec^?1. It was much smaller than that for other kinds of helices, such as an α helix (10¹⁰ sec^?1) or a double helix of nucleic acids (10^7–9sec^?1).     

Polypeptide models of collagen. Solution properties of (Gly-Pro-Sar)n and (Gly-Sar-Pro)n.

Our studies on the solution conformation of (Gly-Pro-Sar)_n and (Gly-Sar-Pro)_n synthesized as polypeptide models for collagen are reported. It is found that, while (Gly-Pro-Sar)_n exists in ordered triple-helical conformation, (Gly-Sar-Pro)_n remains as a disordered random coil in water. Addition of certain helix-promoting solvents seems to generate order in (Gly-Sar-Pro)_n.     

Conformational analysis of polytripeptides (Gly-Pro-Ala)n, (Gly-Ala-Hyp)n,and (Gly-Ala-Ala)n in connection with the problem of collagen structure

Conformational analysis of triple helics of a type of collagen was performed with typical collagen tripeptide sequences based on Gly-Pro-Ala, Gly-Ala-Hyp, and Gly-Ala-Ala. During energy minimization, the possibility of continual deformation of the pyrrolidine cycle was taken into account in order to achieve better accuracy in the resulting structure. The (Gly-Pro-Ala)_n structure is almost isomorphic to the (Gly-Pro-Hyp)_n structure obtained in the previous work [Tumanyan, V. G. & Esipova, N.G. (1982) Biopolymers 21 , 475–497]. For a collagen-type structure, the optimal conformation of (Gly-Ala-Hyp)_n tends to have a decreased unit twist (t = 15°), although the energy advantage with respect to the conformation with t = 45° is not so significant. A similar situation is observed for (Gly-Ala-Ala)_n. In this case, the energy decrease during unwinding to t = 15° from t = 45° is quite small. The conformations of (Gly-Ala-Hyp)_n and (Gly-Ala-Ala)_n with t = 15° exhibit a similarity with a triple complex of polyproline II helices—a noncoiled coil such as (Gly-Pro-Hyp)_n and (Gly-Pro-Ala)_n. A similar structure may be postulated for subcomponent cq1 of the first component of a human complement containing substantial Gly-X-Pro and Gly-X-Y tripeptide derivatives in the primary structure (X, Y = any amino acid). The results suggest that the observed helical symmetry of collagen (t = 36°) is a consequence of superposition of diffraction patterns (for sufficiently long segments) from various helices (t varies from ～15° for Gly-X-Hyp and Gly-X-Y to ～56° for Gly-Pro-Ala). For short alternating segments, some unification of different helical structures is possible.     

Hydroxylation of (Pro-Pro-Gly)5 and (Pro-Pro-Gly)10 by prolyl hydroxylase. Evidence for an asymmetric active site in the enzyme.

Previous studies with 14C-labeled synthetic peptides demonstrated that prolyl hydroxylase, which synthesizes the hydroxyproline in collagen, preferentially hydroxylates the fourth triplet from the NH-terminal end of the peptide (Pro-Pro-Gly)5. In the experiments reported here, the prolyl hydroxylase reaction was investigated further by preparing chemically modified derivatives of (Pro-Pro-Gly)5 and by synthesizing 14C-labeled preparations of (Pro-Pro-Gly)10. Essentially, the same kcat value was found for the hydroxylation of (Pro-Pro-Gly)5, N-acetyl-(Pro-Pro-Gly)5, (Pro-Pro-Gly)5 methyl ester, (Pro-Pro-Gly)10, and for larger polypeptide substrates of the enzyme. It appeared therefore that preferential hydroxylation of specific triplets in peptides of the structure (Pro-Pro-Gly)n cannot be explained by differences in the kinetic constants for individual triplets. Hydroxylation of 14C-labeled preparations of (Pro-Pro-Gly)10 demonstrated that the ninth triplet was preferentially hydroxylated over any other triplet. The results were best explained by the hypothesis that prolyl hydroxylase has an asymmetric active site in which binding subsites are located adjacent to but not symmetrical with the catalytic subsite.     

Polypeptide models of collagen: Properties of (Pro-Pro-betaAla)n.

We have synthesized (Pro-Pro-βAla)_n as a model for collagen. The synthetic polytripeptide, mol wt 6500, exhibits a large negative optical rotation with a very strong negative Cotton effect centered at 216 nm. The optical rotatory dispersion of (Pro-Pro-βAla)_n followed a single-term Drude equation and the λ_c was 195 nm. The rotation decreased markedly on heating with the midpoint of the broad transition at 55°C. Preliminary studies also showed loss of structure in guadinine HCl. The circular dichroism spectrum of the polymer exhibited a deep trough at 190 nm. The marked similarities of solution properties of (Pro-Pro-βAla)_n to (Pro-Pro-Gly)_n suggest that β-alanine can replace glycine in generating collagen-like helix in solution.     

Conformational studies on sequential polypeptides Part VI. Structural investigations on (Pro-Leu-Gly)10, (Pro-Leu-Gly)n and (Leu-Pro-Gly)n.

The conformational properties of (Pro-Leu-Gly)10, (Pro-Leu-Gly)n and (Leu-Pro-Gly)n were investigated both in solution and in solid state. By circular dichroism studies it was possible to demonstrate the formation of an ordered collagen-like structure for (Pro-Leu-Gly)n in hexafluroisopropanol-water mixtures and in ethylene glycol; (Leu-Pro-Gly)n assumes an ordered conformation only in ethylene glycol; (Pro-Leu-Gly)10 is unordered under all the conditions studied. X-ray diffraction patterns indicated that (Pro-Leu-Gly)n and (Leu-Pro-Gly)n assume a triple helical structure in solid state. In addition, the investigation of (Pro-Leu-Gly)n strongly suggests that this type of structure is a single chain triple helix. The X-ray patterns of (Pro-Leu-Gly)10 do not allow us to ascertain a collagen or polyproline II-like structure for this decatripeptide.     

Correlation between Raman and X-ray crystallography data of (Pro-Pro-Gly)10

Model biopolymers are powerful tools to guide the interpretation of physical properties in complex systems. (Pro-Pro-Gly)(10), (PPG)(10), is a collagen-model peptide, whose structure is known at high resolution. Herein, Raman microscopy data of (PPG)(10) powders and single crystals are reported. The spectra interpretation leads to an accurate assignment of three well-resolved amide bands corresponding to the three peptide bonds (PP, PG and GP) present in the (PPG)(10) structure. These data together with the availability of torsional angles phi and psi derived from the high-resolution crystal structure, provide the opportunity to test the validity of theoretical equations for the calculation of non-canonical amide III bands and represent a reference for theoretical calculations of vibrational spectra and for polyproline II detection in complex proteins. Spectroscopic data do not support the indication of two distinct and equally populated up and down conformations of the pyrrolidine rings observed in the (PPG)(10) crystal structure.