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INVESTIGATION OF MOLECULAR STRUCTURE OF THE CORTEX OF WOOL FIBERS Mark I. Liff Philadelphia University phone: 215 951-28

INVESTIGATION OF MOLECULAR STRUCTURE OF THE CORTEX OF WOOL FIBERS Mark I. Liff Philadelphia University phone: 215 951-2879, e-mail: liffm@philau.edu. Introductory Remarks

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INVESTIGATION OF MOLECULAR STRUCTURE OF THE CORTEX OF WOOL FIBERS Mark I. Liff Philadelphia University phone: 215 951-28

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  1. INVESTIGATION OF MOLECULAR STRUCTURE OF THE CORTEX OF WOOL FIBERS Mark I. Liff Philadelphia University phone: 215 951-2879, e-mail: liffm@philau.edu

  2. Introductory Remarks There is a consensus on certain aspects of the structure of wool which was summarized by Fraser (next slide). However, at the molecular level, the structure of wool and especially its interfilament matrix is still not well understood. Elucidation of micro-structure at the molecular level is needed for understanding macroscopic end-use properties of wool. We studied polypeptide models of the matrix phase of wool fibers by 2D 1H liquid state NMR and intact fibers swollen in water and 2H2O by 2H and 1H solid state NMR. Solid state 15N NMR was applied to labeled polypeptide models of the matrix phase. Our experiments have shown that Cys-residues of the dipeptide repeat of the matrix proteins can not serve as cross-links of the network of the matrix phase, while Cys-residues, that are outside of the repeats, can easily perform this function. This explains the elasticity of the matrix.

  3. Suggested by Fraser et. al., CSIRO, www.dwt.csiro.au

  4. The degree of cross-linking of the network has been estimated to be approximately 1 % by application of 15N NMR to the 15N-labeled high sulfur polypeptide of the network. The latter agrees with the known high elasticity of the matrix and its ability to swell in different solvents. A seeming contradiction between high content of Cys-residues in the system (20 %) and low degree of cross-linking (1 %) is shown to be caused by formation of multiple cyclocystine loops upon oxidation. This effectively eliminates the ability of Cys-residues to serve as cross-links of the network. In the intact wool fibers swollen in different solvents, the transverse magnetization and the longitudinal magnetization in the rotating frame revealed a non-exponential decay. Two phases with different sets of NMR-relaxation parameters, T1r (2H), T2 (2H) have been detected. This can be interpreted as a manifestation of the existence of two morphogical phases ofthe cortex.

  5. Formation of a network by different peptide fragments of the HS proteins as the result of oxidation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 B2 protein family S C CQ P T S I Q T S C C Q peptide 1 S C Q P T C LQ T S G C E T G C G peptide 2 T C L Q T S G C E T G C G peptide 3 I C S S V G T C G S S C G Q P T CS peptide 4 The initial linear peptides were oxidized (a) in solution and (b) in solid state in order to obtain a network formed by Cys-Cys disulfide bonding.

  6. MONITORING THE FORMATION OF A NETWORK UPON OXIDATION initial in solution in bulk # 1

  7. The formation of a network manifests itself in the NMR spectra by extreme line broadening seen, for example, in figure 1 • for peptide 2. Spectrum A is for the initial linear peptide, spectrum B is for the peptide oxidized in solution and C for the system obtained by oxidation of peptide 2 in bulk. The lines trace the changes in the chemical shifts. • The results of the oxidation experiments are summarized in the next two figures. They show that • *peptides 3 and 4, which do not contain the repeats in a systematical way, are easily oxidized to a network in both states, in solution and in bulk. • *peptide 1, which contains two dipeptide repeats, does not form a network under both conditions, unlike all other peptides. Its NOESY spectra show that it prefers the formation of two tiny cyclocystine loops to the inter-chain disulfide bonding. The latter precludes the formation of a network.

  8. Summary of the oxidation experiments peptide 1 oxidation in solution S C C Q P T S I Q T S C C QS C C Q P T S I Q T S C C Q + no gel oxidation in solid state for peptide 2 SCQPTCLQTSGCETGCG + no gel oxidation in solution SCQPTCLQTSGCETGCG oxidation in bulk visible gel (network) phase, where stands for a disulfide bond. # 2

  9. Peptide 3 high m.w. fraction (pieces of a network) oxidation in solution T C L Q T S GC E T GC G oxidation in bulk visible gel (network phase) Peptide 4 high m.w. fraction (pieces of a network) oxidation in solution I CS S V G T C G S S C G Q P T CS oxidation in bulk visible gel (network phase) # 3

  10. Solid state 15N NMR of the network. An estimate of the density of cross-linking. Peptide 2*, which is similar to peptide 2, but with 15N for peptidic nitrogen in residue Gln-7, has been synthesized and oxidized in bulk to form a network. In figure 4, the 15N NMR spectrum of a dry sample (A) of the cross-linked peptide 2* represents the expected powder pattern. Line C is for the system swollen to equilibrium in DMSO. The swelling equilibrium ratio of 1:10 is characteristic of a slightly cross-linked polymer. Line C is centered at the isotropic value of 98 ppm and has a width of 2 orders of magnitude greater than for a similar linear peptide. On the other hand, the width of line C is about 5 times smaller than the value of wII - wI = 9000 Hz of line A for the dry sample. The latter suggeststhat that a length of the network’s chain is at least 5 statistical segments, or 50 residues that corresponds to cross-linking at 1 % (consistent with other data).

  11. 15N NMR swollen in DMSO swollen in water dry sample # 4

  12. Relaxation of Water Absorbed by Wool Fibers. • The existence of large-scale phases of more than 100 A, the matrix and the fibrils with different dynamic properties, leaves a possibility of interpretation of the relaxation results in terms of these morphological phases. • Deuterium studies (figure 5) offer advantages over similar proton studies (figure 6). The presence of rigid protein protons (a steep incline in figure 6) with short spin-relaxation times becomes unimportant. The effects of cross relaxation for deuterium should be smaller than for protons. • Deuterium T2-decay (on the figure) was measured by Carr-Purcell-Meiboom-Gill method with the inter-180o pulse delay of 100 ms. The best fit has been achieved with two exponents. • The deuterium T1r experiments also yielded two phases with similar populations. • The analysis in terms of two structural phases, • most likely, oversimplifies a complicated system.

  13. Concluding Remarks * The Cys-Cys repeat, in both states, prefers formation of tiny intra-repeat cyclocystine loops to inter-repeat and inter-peptide bonding. The cysteines of the pentapeptide repeat and other cysteines, on the contrary, show strong propensity for inter-chain bonding leading to the formation of a network. ** The formation of cyclocystines in peptide 1 eliminates potential cross-links of a network, and that makes the formation of a network impossible. This explains high elasticity of the matrix despite a high content (20%) of potential cross-links. *** The average degree of crosslinking for a model polypeptide network is estimated on the basis of the residual 15N CSA interaction to be no more than 1 mol. %. This value fits the description of the matrix as a slightly cross-linked network. **** A multiexponential decay of transverse magnetization and the magnetization in the rotating frame, most likely, reflect the existence of different morphological phases.

  14. ACKNOWLEDGMENTS. • Dr. Ronald McNamara and Dr Kathleen Valentine at the Resource for Solid State NMR of Proteins at the University of Pennsylvania helped with the NMR measurements. • Michael Zimmerman and Bryan Frieman, PCT&S undergraduate students, helped with sample preparations and processing 2D spectra. • REFERENCES : (1) Lindley, H. In Chemistry of Natural Protein Fibers, 1977, p. 147, New York, Plenum. (2) Parry, D. A. D.; Fraser, R. D. B.; MacRae, T. P. Int. J. Biol. Macromol. 1, 1979, 17. (3) Fraser, R. D. B.; MacRae, T.P.; Sparrow, L. G.; Parry, D. A. D. Int. J. Biol. Macromol. 10, 1988, 106. (4) Liff, M.I. Polym. Gels & Networks 4, 1996, 167. (5) Liff, M.I. and Siddiqui, S.S. Int. J. Biol. Macromol. 19, 1996, 139. (6) McLoughlin, K.; Waldbieser, J.K.; Cohen, C.; Duncan, T.M. Macromolecules 30, 1997, 1044. (7) Callaghan, P.T.,;Samulski, E.T. Macromolecules 30, 1997, 113.(8) Cohen-Addad, J.P. Macromolecules 22, 1989, 147; Cohen-Addad, J.P.; Vogin, R. Phys. Rev. Lett. 33, 1974, 940; (9) Gotlib, Y.Y.; Lifshits, M.I.; Shevelev, V.A.; Lishanski, I,S.; Balanina, I.V. J. Polym. Sci. USSR 18, 1976, 2630; Gotlib, Y.Y; Kuznetsova, N.N; Lifshits, M.I.; Papukova, K.P.; Shevelev J. Polym. Sci. USSR B16 1974, 796. (10) Lifshits, M.I. Polymer 28, 1987, 454. (11) Brereton, M.G.; Ries, M.E. Macromolecules 29, 1996, 2644. (12) Gronski, W.; Emeis, D.; Brüderlin, A.; Jacobi, M.; Stadler, R. Brit. Polym.J. 17, 1985, 103; Gronski, W.; Stadler, R.; Jacobi, M. Macromolecules 17, 1984, 741, 19, 1986, 2884. (13) Dickinson, L.C.; Chien, J.C.W.; • MacKnight, W.J. Macromolecules 21, 1988, 2959; 23 1990, 1279.(14) Liff, M.I. • Macromolecules 26, 1993, 551.

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