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.     

Polypeptide models of collagen. II. Solution properties of (Pro-Gly-Phe)n

The conformation of (Pro-Gly-Phe)_n in trifluoroethanol was investigated using CD, nmr and ir techniques. After making appropriate correction for the contribution of the phenylalanine chromophore to the observed CD spectra of the polytripeptide at several temperatures, it is found that (Pro-Gly-Phe)_n can exist in a partially triple-helical conformation in this solvent a t low temperatures. The nmr and ir data support this conclusion. In conjunction with recent theoretical sutdies, our data offer an explanation for the preferential occurrence of the Phe residue in position 2 of the tripeptide sequence Gly-R₂-R₃, in collagen.     

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.     

Two types of tripeptide conformation in collagen. Calculation of the structure of (Gly-Pro-Ser)n and (Gly-Val-Hyp)n polytripeptides

Conformational analysis of polypeptides (Gly-Pro-Ser)n and (Gly-Val-Hyp)n was carried out for collagen-like triple helical complexes (coiled coils with screw symmetry). The lowest energy structure of the first polymer (helical parameters t 52,8, h 0,282 nm) is very close to that of (Gly-Pro-Hyp)n. The hydroxyl group of a serine residue does not form any intramolecular hydrogen bonds in this structure. (Gly-Val-Hyp)n triple complex is shown to unwind to t 7,7, h 0,297 nm as a result of optimization procedure. These findings confirm the assumption, made earlier on the basis of conformational analysis of (Gly-Pro-Hyp)n, (Gly-Pro-Ala)n, (Gly-Ala-Hyp)n, (Gly-Ala-Ala)n, that the collagen triple helix contains stable wound triplets with proline in the second position, while the absence of imino acid in the 2nd position facilitates the unwinding of the triple helix. Thus, a collagen helix appears to have different parameters for the sites differing in the amino acid sequence. The values measured in the X-ray experiments (h 0,29 nm, t' 36) should be considered as a result of averaging. The model allows to reconcile the X-ray data for collagen and crystalline (Gly-Pro-Pro)10 oligomer.     

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.     

The association reaction of collagen model polypeptides (Pro-Pro-Gly)n

The characterization of recently synthesized (Pro-Pro-Gly)_n, n = 7, 8 is described, along with melting profile studies of its association equilibrium, and thermal quenching studies of the kinetics of its association reaction. The order of the kinetic reaction is about 3, implying that three peptide chains are involved in the activated state of the rate-limiting step. The reaction rate was found to exhibit a negative temperature coefficient. With the (Pro-Pro-Gly)₇ peptide, the concentration dependence of the (Pro-Pro-Gly)_n association equilibrium was observed for the first time. Detailed thermodynamic analysis for these n = 7, 8 data, together with literature data for n = 10, 15, 20 were carried out for both the simple “all-or-none” binding model and for a series of complex equilibrium models. For the latter, all of the (Pro-Pro-Gly)_n data (in 10% acetic acid) are fit best with a maximally cooperative near-neighbor model with a standard enthalpy change ΔH = ?650 cal/mole of residues, and a standard entropy change ΔS = ?14.63 ?10/n cal/deg-mole of residues, wherein the ?10 eu represents an end-effect contribution to the binding free energy. With regard to optical rotatory properties and thermodynamic parameters, the data for the new n = 7, 8 peptides match rather well with the literature data for the n = 10, 15, and 20 peptides. The enthalpic stabilization per residue of the triple-helical form of (Pro-Pro-Gly)_n was nearly an order of magnitude smaller than the enthalpic stabilization per additional proline obtained from direct calorimetric measurements on native collagens of different (and much lower) proline contents by Privalov and Tiktopulo. [Biopolymers (1970) 9 , 127–139.] Possible explanations for this phenomenon are discussed.     

植物多肽信号分子的特点和功能

近几年研究表明,植物体内存在类似动物和酵母的多肽信号分子,调控植物生长发育以及对环境的响应.介绍了植物中的系统素、迅速碱化因子(RALF)、早期结瘤蛋白40(ENOD40)、植物磺化激动素(PSK)、S位点富含半胱胺酸蛋白(SCR)、CLV3以及相应受体的特点和功能研究进展,并且对多肽信号在植物中的作用及其应用前景进行了探讨.     

Chromatin models. Thermal denaturation studies of (Lysx, Leuy)n-and (Lys)n(Leu)m-DNA complexes.

The structural transitions of (Lysx, Leuy)n-DNA and (Lysx)n(Leuy)m-DNA complexes have been studied by thermal denaturation utilizing simultaneous absorption and circular dichroism (CD) measurements [R. Mandel and G.D. Fasman (1974), Biochem. Biophys. Res. Commun. 59, 672]. These complexes are used as models for nucleohistones. At amino acid/nucleotide ratios r less than 1, the copolymers bind to DNA in a ratio of one amino acid residue per nucleotide, and such binding stabilizes the DNA double helix against thermal denaturation relative to the unbound regions. The leucine residues in the copolymers stabilize the bound portion of the complex against thermal denaturation but to a lesser degree than does poly(L-lysine). This study confirms the hypothesis that absorption melting profiles reflect only the change in secondary structure (helix-coil transition) of DNA. It was found that, in the absence of a higher ordered structure (condensed), the CD melting profile also reflects this same conformational transition, and the melting temperatures, Tm, in CD are equal to those in absorption. However, when a higher ordered structure (tertiary) exists in the complex, then the CD melting profile will be dominated by the structural transitions related to the melting of the higher ordered asymmetric structure in the condensed state, followed by the melting of the secondary structure. Under such circumstances, the Tm obtained from absorption may be slightly different from that of the CD, since only the secondary structural changes are being reflected in absorption. The relevance of these studies to the structure of chromatin is discussed.     

Polypeptide fluoroacetate derivatives. Synthesis and 19F n.m.r

The synthesis and characterization of monofluoroacetyl (MFAc) functionalized haptens are described. These were covalently bound to polypeptide carriers (bovine serum albumin and poly-D-lysine) by primary amine/succinimide ester or primary amine/acid chloride coupling. Epitopic densities of the resulting antigens were determined by both 19F n.m.r. and picryl sulfonic acid assays. 19F n.m.r. experiments defining the stability of MFAc ester and amide linkages as a function of media (including in vitro), time, and temperature are presented. These results indicate that MFAc functionalized antigens are well suited for further immunologic studies.     

Conserved Properties of Polypeptide Transport-associated (POTRA) Domains Derived from Cyanobacterial Omp85

Proteins of the Omp85 family are conserved in all kingdoms of life. They mediate protein transport across or protein insertion into membranes and reside in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Omp85 proteins contain a C-terminal transmembrane β-barrel and a soluble N terminus with a varying number of polypeptide-transport-associated or POTRA domains. Here we investigate Omp85 from the cyanobacterium Anabaena sp. PCC 7120. The crystallographic three-dimensional structure of the N-terminal region shows three POTRA domains, here named P1 to P3 from the N terminus. Molecular dynamics simulations revealed a hinge between P1 and P2 but in contrast show that P2 and P3 are fixed in orientation. The P2-P3 arrangement is identical as seen for the POTRA domains from proteobacterial FhaC, suggesting this orientation is a conserved feature. Furthermore, we define interfaces for protein-protein interaction in P1 and P2. P3 possesses an extended loop unique to cyanobacteria and plantae, which influences pore properties as shown by deletion. It now becomes clear how variations in structure of individual POTRA domains, as well as the different number of POTRA domains with both rigid and flexible connections make the N termini of Omp85 proteins versatile adaptors for a plentitude of functions.     

Thaumamermis cosgrovei n. gen., n. sp. (Mermithidae: Nematoda) parasitizing terrestrial isopods (Isopoda: Oniscoidea)

Summary A new mermithid nematode, Thaumamermis cosgrovei n. gen., n. sp. (Mermithidae: Nematoda) was found parasitizing two terrestrial isopods (Isopoda: Oniscoidea) in California. The hosts, Armadillidium vulgare (Latr.) (a pillbug) and Porcellio scaber (Latr.) (a sowbug) represent the first cases of isopods attacked by mermithid nematodes. The genus Thaumamermis can be distinguished from all previously described mermithids by the extremely dimorphic spicules, one being short and broad and the other long and filiform. It has been discovered that the nematodes are infected with an iridiovirus which commonly destroys the isopod hosts. ac]19800917