Investigation of Molecular Structure of the Cortex of Wool Fibers

Transcription

1 Investigation of Molecular Structure of the Cortex of Wool Fibers (Seed Project) Principal investigator: Mark Liff, Philadelphia University Participants: Ronald McNamara, University of Pennsylvania Michael Zimmerman, undergraduate student, Philadelphia University Bryan Frieman, undergraduate student, Philadelphia University Goal A fundamental understanding of the relationships between the molecular structure of wool fibers and their "end-use" macroscopical properties, such as flexibility and elasticity, is needed to foster novel ideas for synthesis of new fibers with the properties that are similar or better than those of natural fibers. Among the objectives of this project is the elucidation of the structure of the major components of the cortex of wool fibers, the matrix phase and the intermediate filaments, as well as the elucidation of how these two phases are linked together to form a fiber. Abstract Polypeptide models of the matrix phase of wool were studied by high resolution liquid state 1 H NMR, and intact wool fibers swollen in water and D 2 O were studied by relaxation 1 H and 2 H NMR which is often performed in solid state. A network structure of the matrix phase was modeled by polypeptide systems containing fragments of high sulfur (HS) proteins from wool with Cys-residues oxidized in pairs to form a network. The oxidation of three- and four-cys-residue peptide models of the matrix has been monitored by 2D 1 H NMR. Two peptides, TCLQTSGCETGCG and ICSSVGTCGSSCGQPTCS, which do not contain the characteristic dipeptide and the pentapeptide repats, have been assigned in the initial reduced state, and then analyzed in the oxidized state. The results of these studies suggest that Cysresidues outside the dipeptide and the pentapeptide repeats can serve as cross-links of a network formed by oxidation under both conditions studied, in solution and in bulk, that is different from the behavior of Cys-residues in the dipeptide and pentapetide repeats studied earlier. The existence of several types of Cys-residues in terms of their propensity to serve as cross-links in the HS protein systems explains the possibility for formation of a slightly cross-linked network in a polypeptide system which is extremely rich (up to 20 %) in Cys-residues, a potential cross-linking agent. In wool fibers swollen in water and D 2 O, the transverse magnetization and the spin-locked magnetization of the solvents revealed a nonexponential decay. Two NMR-phases with different sets of NMR-relaxation parameters, T 1ρ ( 2 H), T 2 ( 2 H), have been detected: the values of these NMR parameters are in the intermediate region between liquids and solids. These data may suggest that different structural phases of the fiber contribute to the net signal. 1

2 Introduction There is a consensus that the cortex of wool fibers includes semi-crystalline microfibrils imbedded into the amorphous elastic interfilament matrix. 1-5 Cys-residues of high sulfur (HS) proteins of the matrix are oxidized in pairs to give a network with disulfide bridges as cross-links. Cys-residues abound in the HS protein sequences which are dominated by two repeats--a dipeptide Cys-Cys (two Cys are in the adjacent positions on a chain) and a pentapeptide Cys-Xxx-Pro-Yyy-Cys (where Xxx and Yyy are frequently Gln and Thr, respectively). In our previous works 6-8 we have shown that Cys-residues in both repeats have a propensity to form intra-repeat loops, and that this propensity is exceptionally high for the dipeptide repeat. We have also discussed that the formation of an intra-repeat loop should effectively eliminate its two Cys-residues as potential half-cross-links of a network. The latter explains the known properties of the matrix and reconciles a controversy between the high number of oxidized Cys-residues in the matrix of approximately 20% and its high elasticity that is typical for a slightly cross-linked network of an order of 1% of a cross-linking agent or less. However, it is not known what Cys-residues perform the function of cross-links in a weakly crosslinked network of the matrix. To help to answer this question we present here the results of studies of peptides from HS proteins that contain only those Cys-residues that do not belong to the repeats. We studied two peptides, TCLQTSGCETGCG, peptide 1, res in the B2 family of proteins, and ICSSVGTCGSSCGQPTCS, peptide 2, res from the same sequence. Following the approach developed earlier 6-8, we oxidized Cys-residues of these peptides under two sets of conditions, in dilute solution (to avoid inter-chain contacts) and in bulk (to introduce inter-chain contacts). 1 H NMR was used to monitor changes upon oxidation. The most important result of these studies is that Cys-residues outside the repeats, unlike Cys-residues of the repeats, form inter-chain disulfide bonds both in solution and in bulk, suggesting that Cys-residues outside the repeats can serve as cross-links of the matrix network. In an attempt to monitor simultaneously the microfibril phase and the matrix phase in the intact fiber we studied proton and deuterium relaxation of wool swollen in water and in D 2 O. The existence of large on molecular scale (100 A and more) phases, the matrix and the fibrils, with different dynamic properties leaves a possibility of interpretation of the relaxation results in terms of these structural phases. Deuterium studies offer several important advantages over similar proton studies. The presence of rigid protein protons with short spin-relaxation times becomes unimportant. The effects of cross relaxation for deuterium should be smaller than for protons. A multi-exponential decay of transverse magnetization and spin-locked magnetization of protons and deuterons was observed in the solvents absorbed by wool fiber. These data could imply that different structural phases of the fiber (not necessarily from the cortex) contribute to the net signal. Experimental 2

3 Peptides. A 13-residue peptide 1 and a 18-residue peptide 2 from the sequences of the HS proteins of the matrix, Ac- T C L Q T S G C E T G C G NH 2 (peptide 1, res , B2 proteins) Ac- I C S S V G T C G S S C G Q P T C S- NH 2 (peptide 2, res , the same proteins), were synthesized and purified (more than 90 % pure by HPLC) and characterized by LC-MS and HPLC by Multiple Peptide Systems, Inc. (San Diego, CA). The primary structure of peptides 1 and 2 was consequently confirmed by our NMR experiments. Oxidation. Each of peptides 1 and 2 were oxidized in two different ways--in solution and in bulk. In the first case oxygen was slowly bubbled into 5 mm solution of a peptide in DMSO (the peptides are not soluble in water) for 24 hr. at 25 o C. The oxidation was performed directly in the NMR tube, and then NMR spectra of the unpurified sample were taken. The completion of the oxidation was verified by the absence of the NMR signals for the reduced form. For the oxidation in bulk the polypeptide system was slightly swollen in DMSO (or water) at mg of peptide in µl of the solvent and kept in the oxygen atmosphere for 48 hours. Samples. The samples for liquid state 2D NMR studies were approximately 5 mm of a peptide in DMSO-d 6. To maintain a similar molar concentration for different samples needed for comparison, the prepared solution has been divided into two or three equal parts which were placed into separate NMR tubes. One sample was kept unchanged and used as a control sample, the other ones were oxidized under different conditions (or first dried, then oxidized). For solid state 2 H NMR studies merino wool was initially bone -dried and then swollen to equilibrium in D 2 O. The content of D 2 O at equilibrium was determined gravimetrically at approximately 12%. NMR spectroscopy. For liquid state 2D experiments incremental FIDs of 1K complex points were collected in a phase sensitive mode. For TOCSY or clean-tocsy 9 the length of the soft 90 o pulse was approximately 20 ms. The MLEV mixing time was approximately 50 ms. NOESY at 200 ms mixing time was employed for the DMSO-d6 solutions; all NOEs were negative. TOCSY, DQF-COSY and NOESY pulse sequences were used for assignment. 10 A signal of the residual water was presaturated in all spectra. For 2D spectra the data were zero-filled to 1K before the second Fourier transform. Shifted cosine bell and skewed cosine bell functions were used for apodization. The solid state NMR experiments were performed at the University of Pennsylvania Solid State NMR Resource on a home-built double resonance NMR-spectrometer operating at the 1 H NMR frequency of 550 MHz. The decay of proton and deuterium transverse magnetization was measured by Carr-Purcell-Meiboom-Gill method with the inter-180 o -pulse delay of 200 µs for deuterons and protons. 3

4 Spin lattice relaxation was studied by application of inversion-recovery quadrupole echo for deuterons and 180 o -90 o pulse sequences for protons. Results and discussion 1 H NMR of peptides 1 and 2 and the propensity of their Cys-residues to serve as cross-links of the matrix network. The assignment of 1 H spectra of peptides 1 and 2 by conventional NMR techniques -- TOCSY, 2QF-COSY and NOESY-- was straightforward, and is presented in tables 1 and 2. Sharp differences between the initial linear peptides 1 and 2 and the systems obtained as the result of their oxidation can be seen in the amide regions of their 1D NMR spectra (Figures 1 and 2). The initial linear peptide 1 (spectrum 1A) is compared to the peptide systems obtained by oxidation of peptide 1 in solution (spectrum 1B) and in bulk (1C). The three samples have similar initial amount of peptide 1 -- the oxidation in solution and the oxidation in bulk have been performed directly in the NMR tubes, and no peptide has been added (or removed) in the process to any of the three tubes. The spectra were obtained under the identical spectroscopic conditions. However, the samples were not similar in terms of their homogeneity. The solutions of the initial peptide 1 was transparent and did not contain a visible precipitate, while the attempts to dissolve the peptide oxidized in bulk in different solvents (DMSO, methanol, chloroform, water at different ph) were not successful. The difference in the phase composition of the samples is reflected in the appearance of their NMR spectra. While spectrum A is well resolved and its line intensity is normal for linear peptides at similar concentrations, the intensity of spectrum B and, especially, C are low -- the signals practically vanish from spectrum C. This is in agreement with the existence of a swollen network phase having lines too broad to be observed and assigned by liquid state NMR. The conversion to a gel (network) phase, as a result of the oxidation of peptide 1 in bulk and in solution, should occur at a high yield since the signals of the peptide remaining in the solvent phase are broad and small. The latter can be concluded from a comparison of spectra C and B with spectrum A. A network structure is expected to be more continuous for the case of oxidation in bulk that is reflected in broader and smaller peaks for spectrum C. The behavior of peptide 2 (figure 2) is similar to that of peptide 1. The results of the oxidation experiments for peptides 1 and 2, suggest that their Cys-residues prefer intermolecular disulfide bonding that leads to a gel phase formation under both conditions of oxidation, in solution and in bulk. This is quite different from the results obtained earlier under similar conditions for polypeptides containing the dipeptide and pentapeptide repeats. 6-8 In solution, Cys-residues of the repeats prefer formation of intra-repeat disulfide bonds to formation of inter-molecular disulfide bonds leading to a network structure. Cys-residues of the dipeptide repeat maintain the same preference even in the case of oxidation in bulk. The presented data suggest that nonrepeat Cys-residues can serve as cross-links of the matrix network while Cys-residues of the dipeptide 4

5 repeat and pentapeptide repeat (only under the oxidation-in-solution conditions 6 ) can be effectively eliminated as potential cross-links due to their participation in the formation of intra-repeat loops. The fact that only a small fraction of Cys-residues in the HS proteins is outside of the repeats, suggests that most of the potential cross-links of the network can be eliminated by oxidation. Therefore, relatively few Cys-residues can serve as cross-links of the network that can explain high elasticity of the matrix and its ability to swell in solvents despite a high content (20%) of the potential cross-linking agent (Cysresidues). It should be mentioned that for oxidation of some hypothetical enzymatic nature, the results could be quite different from those obtained here for oxidation by air. Relaxation of water and D 2 O absorbed by wool. Moisture regain in wool under normal conditions is approximately %. The distribution of water among different phases of wool fibers is not known, and the dynamics of water molecules in the fiber has not been studied. The interpretation of water relaxation experiments in water-macromolecule systems in terms of differences in molecular surroundings is complicated by cross relaxation phenomena and other factors. In case of wool the existence of large on molecular scale (100 A and more) phases with different dynamic properties (the matrix and the fibrils) leaves a possibility of interpretation of the relaxation results in terms of these different morphological phases. A signal of H 2 0, or D 2 O in wool contained only one broad bell-shaped peak which is neither lorenzian, nor a superposition of lorenzians. The proton line width was 3600 Hz and for deuterium the width was 800 Hz. Single exponents fit the proton and deuterium inversionrecovery curves. The decay of proton transverse magnetization (figure 3) exhibits at least two phases with a difference in the values of spin-spin relaxation times of two orders of magnitude. In this case, the manifestation of two components of the cortex might be obscured by the contribution from the rigid protons of the proteins. Deuterium studies offer several important advantages over similar proton studies. The presence of rigid protein protons with short spin-relaxation times becomes unimportant. The effects of cross relaxation for deuterium should be smaller than for protons. The best fit for the decay of deuteron transverse magnetization (figure 4) has been achieved with two exponents with T 2a =.55 ms, T 2b = 1.85 ms, with population of phase a at approximately 85 %. The deuterium T 1ρ experiments (figure 5) also yielded two phases with similar differences in the values of the relaxation parameters that may correspond to two different structural phases of the fiber, though not necessarily those of the microfibrils and the matrix. It should be also mentioned, that the analysis in terms of two structural phases only, would, most probably, oversimplify a very complicated system. Conclusion. Cys-residues outside the dipeptide and pentapeptide repeats of high sulfur proteins of the matrix have a propensity to serve as cross-links of a network formed as a result of oxidation in bulk and in solution. This is different from the behavior of Cys-residues of the repeats studied under similar 5

6 conditions earlier. The existence of several types of Cys-residues in terms of their propensity to serve as cross-links of the matrix network explains the elasticity of the matrix and its ability to swell in solvents. In the intact wool fibers swollen in water and D 2 O, transverse magnetization and longitudinal magnetization in the rotating frame revealed a non-exponential decay. At least two phases with different sets of NMR-relaxation parameters have been detected in T 1ρ ( 2 H) experiments and in T 2 ( 2 H) experiments. These data could imply that different structural phases of the fiber contribute to the net signal. 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) Feughelman, M. Keratin, Encycl. Polym. Sci. Eng., 1988, p.566. (5) Marshall, R.C. In Proceedings of the 8th International Wool Reasearch Conf., 1990, p. 169, Christchurch, New Zealand. (6) Liff, M.I. and Zimmerman, M.N. Polym. Internat. 47, 1998, 375. (7) Liff, M.I. Polym. Gels & Networks 4, 1996, 167. (8) Liff, M.I. and Siddiqui, S.S. Int. J. Biol. Macromol. 19, 1996, 139(9) Griesinger, C.; Otting, G.;Wüthrich, K.; Ernst, R.R. J. Amer. Chem. Soc. 110, 1988, 7870.(10) Wüthrich, K. NMR of Proteins and Nucleic Acids, 1986, New York, Wiley. Figure 1. 1 H NMR spectra of (A) the initial linear peptide 1, (B) the system obtained by oxidation of peptide 1 in solution and (C) the system obtained by oxidation of peptide 1 in bulk. Each of the three samples had similar initial amount of the peptide, approximately 5 mm in DMSO. The spectra are obtained under similar spectroscopic conditions. 6

7 Figure 2. 1 H NMR spectra of (A) the initial linear peptide 2, (B) the system obtained by oxidation of peptide 2 in solution and (C) the system obtained by oxidation of peptide 2 in bulk. For experimental conditions see Experimental and the caption to figure 1. Figure 3. Decay of proton transverse magnetization of merino wool with absorbed water. Bi-exponential fit: A = A a * exp(-t/t 2a ) + A b * exp(-t/ T 2b ), A a : A b = 25 : 10, T 2a =.2 ms and T 2b = 20 ms. 7

8 Figure 4. Decay of deuteron transverse magnetization of D 2 O absorbed on wool. Bi-exponential fit: A = A a * exp(-t/t 2a ) + A b * exp(-t/ T 2b ), A a : A b = 62 : 10, T 2a =.55 ms and T 2b = 1.85 ms. Figure 5. Decay of deuteron spin-locked magnetization of D 2 O absorbed on wool. Bi-exponential fit: M = M a * exp(-t/t 1ρ a ) + M b * exp(-t/ T 1ρ b ), A a : A b = 73 : 10, T 1ρ a =.95 ms and T 1ρ b = 5 ms. 8

9 Table 1. Chemical shift assignments for peptide 1 in DMSO-d 6, t = 24 o C. Thr 1 Cys 2 Leu 3 Gln 4 Thr 5 Ser 6 Gly 7 Cys 8 Glu 9 Thr 10 Gly 11 Cys 12 Gly 13 NH α β β γ 1, δ δ Table 2. Chemical shift assignments for peptide 2 in DMSO-d 6, t = 24 o C. Ile 1 Cys 2 Ser 3 Ser 4 Val 5 Gly 6 Thr 7 Cys 8 Gly 9 Ser 10 Ser 11 Cys 12 Gly 13 Gln 14 Pro 15 Thr 16 Cys 17 Ser 18 NH α β β γ γ δ 1,