STRUCTURAL AND DYNAMIC INFORMATION FROM MULTIPLE ALIGNMENTS AND CROSS-CORRELATED RELAXATION
W. PETI, J. MEILER, M. REESE, L. VERDIER, S. BECKER, T. CARLOMAGNO, C. GRIESINGER
Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany
Multiple alignments can be used to describe protein dynamics. Two different approaches have been proposed in the literature. Tolman et al. (1,2) used several couplings in a single as well as in multiple alignments. Our approach relies on the measurement of each dipolar coupling in at least 5 different alignment media. It has so far been applied to NH vectors in 11 different alignment media (3). Findings on ubiquitin will be discussed.
Paramagnetic tagging is an alternative to orient diamagnetic proteins without the need for external alignment media. In addition to dipolar couplings, pseudeocontact shifts and Curie/dipole-dipole cross correlated relaxation can be observed. Results of this approach will be shown on ubiquitin as well as triggerfactor will be shown (4).
A third application uses cross correlated relaxation to study structures of intermediately bound molecules (5).
OPTICAL SPECTROSCOPIC PROBES OF UNFOLDED PEPTIDES AND PROTEINS, A PARTIALLY ORDERED POLYMER PROBLEM OF BIOLOGICAL SIGNIFICANCE
T.A. KEIDERLING
Department of Chemistry, University of Illinois at Chicago, 845 W. Taylor St.(m/c 111), Chicago, IL 60607-7061 USA <tak uic.edu>
Proteins are hetero-sequence polypeptides that have complex folded structures whose topology and structural details are vital to their biological function. Thus study of how the fold develops and the energetics and dynamics of attaining that fold for both peptides and proteins has become a vibrant area of research in biophysics. The protein native state is often assumed to be a unique, low energy minimum on the potential surface. Unfolded proteins and unstructured peptides are obviously a vital thermodynamic state in the overall protein-folding problem. They represent one of a possible set of initial states in the folding process (or final states in the unfolding path). However, the disordered species, as attained by various physical and chemical means of denaturing proteins or unfolding peptides, are intrinsically poorly defined and do not represent unique thermodynamic states. In fact data is developing to show these "unfolded" proteins often have considerable local order [1].
Most physical techniques commonly used for protein conformational studies have an analytical response that changes with structure. If different residues are in a variety of local conformations, but these are the same in each molecule, and the technique can differentiate them from a different ensemble of conformations, much structural insight can be gained and folding pathways can potentially be followed. However the unfolded state violates this basic premise in that any given residue in a protein or peptide may be in several different local states. This can cause the technique to have a completely non-systematic response to conformational change, depending on the physical interactions that it exploits. This can pose a problem for local, structurally sensitive techniques such as x-ray and nmr. On the other hand, optical techniques, with much less resolution can provide averaged structure information, which, while not site specific, does represent the ensemble of structures present. Furthermore the time scale of optical spectroscopic probes allows monitoring of fast dynamic process involving these unfolded species.
In this talk, uses of electronic circular dichroism (ECD) in the uv, infrared (IR) absorption and its chiroptical variant, vibrational circular dichroism (VCD) for study of peptide models and unfolded proteins are addressed. The complementary information gained from analysis of the short range vibrational coupling (with IR and VCD) vs. long range dipole coupling (from ECD) will be discussed. The vibrational techniques show evidence of local order often missed by ECD (or fluorescence). Model peptide studies using isotopic labeling for site-specific sensitivity and theoretical analysis will be used to demonstrate unfolding processes for small peptides. Example applications of ECD, IR and VCD in folding and unfolding studies will be presented.
[1] "Unfolded Proteins" Ed. G. D. Rose, Adv. Prot. Chem., 62, 2002 (Academic Press, New York)
LOCAL ORDER IN POLYCARBONATE GLASSES
R. O'CONNOR,a T. WELDEGHIORGHIS,a B. POLIKS,b K. WOOLEY,a J. SCHAEFERa
aDepartment of Chemistry, Washington University, St. Louis, MO 63130.
bDepartment of Physics, Binghamton University, Binghamton, NY 13902
Interchain packing in 13C, 2H, and 19F-labeled polycarbonates has been characterized by 13C{2H} and 13C{19F}rotational-echo double resonance (REDOR) NMR on a 1-2 nm distance scale. Differences in the REDOR dephasing rates of the centerband and spinning sidebands of the carbonyl-carbon resonance indicate local orientational order. The orientational angles are extracted directly from sideband intensities using collections of contour maps for the centerband and each sideband as a function of the REDOR evolution time. This approach is analogous to the use of the familiar Herzfeld-Berger maps to characterize chemical-shift tensors from sideband intensities. The attractive features of this REDOR experiment as a general method for determination of specific orientation in polymers are that it is: (i) one dimensional and hence high sensitivity; (ii) chemical-shift specific because of magic-angle spinning; (iii) accessible to any site that can be stable-isotope labeled; (iv) adaptable to clusters of two or more labels; and (v) suitable for simple spin counting for quantitative analysis. The 13C{19F}REDOR experiments involved a blend of 1% CF3-labeled polycarbonate (the isopropylidene has one CF3 and one CH3) with 99% 13C(=O)-labeled polycarbonate. The observed dephasing is greater than 1% after less than 3 msec dipolar evolution, which indicates that every CF3 has at least one carbonate label within 5 Angstroms. This result is consistent with local pair-wise order in chain packing for bisphenol-A polycarbonate, and inconsistent with random packing.
MOLECULAR ORIENTATION IN ELASTOMERIC NETWORKS BY FOURIER TRANSFORM INFRARED DICHROISM
L. BOKOBZA
Laboratoire de Physico-Chimie Structurale et Macromoléculaire,
ESPCI, 10 rue Vauquelin, 75231, Paris Cedex 05 (France)
Liliane.Bokobza espci.fr
Molecular orientation studies of polymeric systems are of particular interest since they provide valuable information on the mechanisms involved in polymer deformation. Infrared dichroism, which does not require any chain labeling, is one of the most useful tools for a quantitative measurement of this orientation. It measures directly the orientation of specific vector directions within chain segments and thus probes the orientation behavior of network chains on a segmental scale.
This work will illustrate the application of Fourier-transform infrared spectroscopy to the determination of the molecular orientation parameters in some elastomeric networks, the orientation being suitably induced by stretching the sample uniaxially. New developments in the evaluation of segmental orientation such as the polarization-modulation approach and the near-infrared spectroscopy will be discussed.
The strain dependence of segmental orientation will be analyzed through networks of known degree of cross-linking. The experimental results will be compared with calculation predictions based on the rotational isomeric state formalism and some results will be shown on bimodal systems. By deuterating one type of chain, infrared spectroscopy is able to characterize independently the orientation function of each species of a bimodal network consisting of short and long chains. The phenomenon of strain-induced crystallization in natural rubber as well as the evaluation of the orientation of the different chemical sequences of a copolymer will also be illustrated.
Elastomers, particularly those that cannot undergo strain-induced crystallization, are generally compounded with a reinforcing filler such as silica, carbon black or clay in order to improve the mechanical properties of the final materials. The reinforcement of elastomers is probably one of the most important processes in rubber industry and essentially in modern tire technology. The orientational order generated in uniaxially elongated networks is determined by the average number of segments between chemical and physical network junctions. Measurements of chain orientation in filled systems can be used to estimate the total network density arising from the permanent chemical cross-links and from polymer-filler attachments. By quantifying the interaction between the two phases, the analysis of chain orientation provides a new area for understanding the molecular origin of the reinforcement effect.
TIME-RESOLVED VIBRATIONAL SPECTROSCOPY OF STRUCTURAL CHANGES IN SOLID POLYMERS UNDER EXTERNAL PERTURBATIONS
G.G. HOFFMANN, E. KLIMOV, O. KOLOMIETS, I. ZEBGER, H.W. SIESLER
Department of Physical Chemistry, University of Duisburg-Essen, D 45117 Essen, Germany, hw.siesler uni-essen.de
Vibrational spectroscopic techniques (mid-infrared (IR), near-infrared (NIR) /1/ and Raman) are important tools for the analysis of the chemical composition and the characterization of physical phenomena of polymers. Over the last decade new developments in hard- and software have tremendously expanded the applicability of these methods and their information content. Furthermore, the increase in time-resolution down to the microsecond range has opened up novel research areas by offering new insights into very fast processes. Thus, in studies of liquid-crystal display cells and photoaddressable polymers, the reorientation of liquid crystals and liquid-crystalline polymers under the influence of external electric and electromagnetic fields, respectively, has been monitored by time-resolved Fourier-Transform (FT)-IR spectroscopy /2/.
FT-Raman, FT-IR and FT-NIR spectroscopy have also been successfully combined with mechanical measurements to provide a more detailed picture of deformation and relaxation phenomena in socalled rheo-optical experiments of polymers /3/.
The presentation of selected results with reference to the above topics and developments will illustrate the challenge of vibrational spectroscopy for the study of polymer physical phenomena.
/1/ H. W. Siesler, Y. Ozaki , S. Kawata and H. M. Heise (eds.)
Near-Infrared Spectroscopy, Wiley-VCH, Weinheim (2002)
/2/ H. W. Siesler et al., in Modern Polymer Spectroscopy (G.
Zerbi ed.)
Wiley-VCH, Weinheim, pp 33-85 (1999)
/3/ A. K. Kalkar, H. W. Siesler, I. Zebger, F. Pfeifer, A.
Ameri, S. Michel and
U. Hoffmann, in Handbook of Vibrational Spectroscopy (J. Chalmers
and P.R. Griffiths eds.), John Wiley & Sons Ltd., Chichester,
UK, pp 2559-2575 (2002)
DIFFUSIONAL BEHAVIOR OF ROD-LIKE POLYPEPTIDES IN THE LIQUID CRYSTALLINE PHASE BY HIGH-FIELD GRADIENT NMR
I. Ando
Department of Chemistry and Materials Science, International
Research Center
of Macromolecular Science, Tokyo Institute of Technology, 2-12-1
Ookayama, Meguro-ku, Tokyo 152-8552, Japan
The self-diffusion coefficients (D) of rod-like poly(n-alkyl L-glutamate)s having n-dodecyl side chains(PDLG) in the thermotropic liquid crystalline phase have been measured as a function of the main-chain length [molecular weight (Mw) of 7,000, 30,000 and 130,000, which are corresponding to the main chain lengths (L) of ca.30, 200 and 890 Å, respectively)] within the temperature range from 50 to 80 oC by means of high field-gradient 1H NMR method, in order to elucidate the diffusional behavior of the polypeptides in the thermotropic liquid crystalline phase(1-3).
From the experimental results, it is found that at temperatures above the melting point of side-chain crystallites in poly(n-alkyl L-glutamate) the polypeptide forms the thermotropic liquid crystalline state, and then the isotropic diffusion coefficients (Diso) of the rod-like polypeptides are decreased with an increase in the main-chain length.
The diffusion process has been analyzed by the Kirkwood theory of diffusion process for rod-like polymers.
The diffusion coefficients of poly(L-glutamates) in the directions parallel (D∥) and perpendicular (D⊥) to the -helical axis have been determined, and the D∥ value is found to be larger than the D⊥ value.
Further, the self-diffusion coefficients of rod-like PDLG in the lyotropic liquid crystalline phase with chloroform as solvent have been elucidated by high field-gradient NMR(4).
References
FOURIER-TRANSFORM RAMAN SPECTROSCOPY AND WIDE-ANGLE X-RAY DIFFRACTION STUDIES OF MOLECULAR STRUCTURE, CRYSTALLINITY, AND MORPHOLOGY OF UNCOMPATIBILIZED AND COMPATIBILIZED BLENDS OF POLYETHYLENE/NYLON 12
H. SATO1, S. SASAO2, K. MATSUKAWA2,
Y. KITA2, H. YAMAGUCHI1,
H.W. SIESLER3, Y. OZAKI1
1School of Science and Technology, Kwansei Gakuin University, Gakuen, Sanda, Hyogo 669-1337, Japan
2Plastics Department, Osaka Municipal Technical Research Institute, Morinomiya, Joto-ku, Osaka 536-8553, Japan
3Department of Physical Chemistry, University of Duisbrug-Essen, D45117, Essen, Germany
The present study is aimed at investigating differences in molecular structure, crystallinity and morphology between uncompatibilized and compatibilized blends of high-density polyethylene (HDPE) and Nylon 12 by using Fourier-transform (FT) Raman spectroscopy, wide-angle X-ray diffraction (WAXD), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Uncompatibilized and compatibilized blends of HDPE/Nylon 12 with a Nylon 12 content ranging from 10 to 90 wt.-% at an increment of 10 wt.-% were prepared. The compatibilized polymer blends were prepared by adding a small amount of maleic anhydride (MAH) and it was found that 0.5 wt.-% MAH yielded a good dispersion. SEM images show that the uncompatibilized and compatibilized blends have a different miscibility behavior. The uncompatibilized and compatibilized blends yield quite different X-ray diffraction patterns; the latter blends with a Nylon 12 content >70 wt.-% show orientational effects in the X-ray pattern of the HDPE. The crystallinity of the HDPE of both blends was evaluated by the full-width at half intensity of the (110) reflection of HDPE. To do that, the diffraction peaks were analyzed by a curve-fitting method. To evaluate the crystallinity from Raman spectra the intensity ratio of the two bands at 1129 and 1110 cm-1 was used. Of note is that the 1129cm-1 band is due to a symmetric C-C stretching mode of all-trans -(CH2)n- groups arising only from HDPE. The Raman spectra and X-ray diffraction measurements revealed that when the Nylon 12 content reaches 70 wt.-%, the crystallinity of HDPE in the compatibilized blends becomes higher than that of HDPE in the uncompatibilized blends. This result is different from the general trend of crystallinity of HDPE in polymer blends. The difference suggests that the effect of the high viscosity of the Nylon rich phase on the crystallinity is more significant than the effect of the impurity (MAH-grafted PE). It seems that the extension of the Nylon 12 rich phase during the extrusion process due to the increase in the interaction of the MAH-HDPE leads to orientational effects.
NMR MICROSCOPY OF ANISOTROPIC POLYMERS, INCLUDING IMAGING OF EMBEDDED SOLVENT AND OF THE POLYMER ITSELF
D. CANET
Laboratoire de Méthodologie RMN, Université Henri Poincaré, Nancy 1, Faculté des Sciences, B.P. 239, F-54506 Vandoeuvre-les-Nancy, France
A simple experiment, based on radio-frequency field gradients, yields accurate T2 images in a measuring time roughly twice that required for a conventional spin density image. We have performed this experiment on a solvent (toluene) inside polymeric samples of industrial interest, high-density polyethylene (hdpe), and shown that, although the images themselves do not exhibit any correlation with the structural properties of the material, the T2 distribution over all the voxels provides interesting information. When samples are homogeneous, either naturally or subsequently to swelling processes, this distribution is essentially accounted for by a single gaussian function. Conversely, for an ”anisotropic” sample, two gaussian functions are necessary, one of them corresponding to more organized regions of the sample. These features are confirmed by spin density images, which monitor toluene penetration into the material. Solvent diffusion in both samples (isotropic and anisotropic) could be successfully modeled as demonstrated by the comparison between experimental NMR images and simulated images.
Chemical shift imaging can be easily adapted to high-resolution solid-state experiments by means of radio-frequency field gradient technology (the gradient is delivered by a two-turn flat coil, the axis of which coinciding with the rotor axis). The resulting two-dimensional diagram involves chemical shift information in one dimension and spatial information (along the rotation axis) in the other dimension. Different procedures (physical filters) have been considered in order to enhance specific properties of the crystalline and amorphous components in anisotropic hdpe (see above). Two of them rely on differences in cross-polarization or in cross-polarization inversion processes, while a third one is based on carbon-13 longitudinal relaxation times. The latter is shown to be especially efficient for effectively obtaining separate images of the amorphous and crystalline components and ultimately unraveling their distribution within the sample. In particular, it is demonstrated that two structurally distinct amorphous phases are spatially separated in the sample under investigation.
STUDY OF MOLECULAR ORIENTATION BY VIBRATIONAL SPECTROSCOPY: FROM POLYMERS TO SILK
M. PÉZOLET
Centre de recherche en sciences et ingénierie des macromolécules, Département de chimie, Université Laval, Québec, Québec, Canada G1K 7P4
Molecular orientation is generally introduced in natural and synthetic macromolecules by the mechanical deformation that occurs during their processing. Since molecular orientation strongly affects the physical properties of macromolecular systems, it is important to understand the mechanisms that govern the orientation and the relaxation of orientation in such systems. Infrared and Raman spectroscopies are very efficient techniques to characterize the molecular orientation since they can provide information about the degree of orientation of different chemical groups or phases in multicomponent systems such as semicrystalline polymers and copolymers. Infrared linear dichroism (IRLD) has been widely used to study polymer deformation, but its use is limited to relatively slow processes. To improve the sensitivity of this technique and to be able to follow accurately the dynamics of orientation and relaxation, FTIR spectroscopy has been coupled with the polarization modulation (PM) technique. With this technique, the dichroic difference spectrum is recorded directly, thus minimizing instrumental and sample fluctuations. Raman spectroscopy using a confocal microscope is also a powerful technique to characterize the orientation of macromolecules. The main advantage of this technique is that the laser beam can be focused down to about 1 m in diameter in the sample therefore allowing the in situ recording of high quality spectra of single fibers. In addition, the second and fourth terms, <P2> and <P4>, respectively, of the Legendre polynomial expansion of the orientation distribution function can be determined from polarized Raman spectra. In this presentation, examples of the use of PM-IRLD to study in situ the dynamics of orientation and relaxation of stretched semicrystalline polymers and polymer blends will be presented. In addition, results on the use of Raman microspectroscopy to establish structure-property correlations in different silk monofilaments of both spiders and silkworms will be presented. Spider silk is among nature’s most highly engineered structural materials, achieving, in some case, combinations of strength and toughness that could not be reproduced by artificial means. The results obtained by Raman microspectroscopy show that the -sheet domains are highly oriented in spider dragline silk and that in regenerated silk fibers and films of the silkworms Samia cynthia ricini and Bombyx mori, a strain-induced transition from either random coil or -helical conformation to -sheet structure occurs during the mechanical deformation.
NMR investigation of the main-chain orientation in liquid-crystalline side-chain polymers
H. Schneider, G. Hempel
Martin-Luther University, Dept. of Physics, Friedemann-Bach-Platz 6, D-06108 Halle/Saale, Germany
schneider physik.uni-halle.de; hempel physik.uni-halle.de
The structure of the mainchains influences essentially the macroscopic behavior of side-chain polymers, like glass-transition and mechanical as well as rheological properties. Thus their characterization is a basis for establishing structure-property correlations. If the sidechains form liquid-crystalline phases, the mainchains are forced to accept also a certain degree of order.
For a series of mesogenic polysiloxanes, the orientation degrees of the main-chain segments were estimated by 29Si solid-state NMR. It could be concluded that the mainchains are oriented perpendicularly to the side-chain director; only in the case of steric hindrance also parallel arrangement of side- and main-chains was observed. This is a certain discrepancy to conclusions from SANS measurements, which however were performed at a different length scale.
For main-chain segments with laterally attached side-chains another structural parameter will be important: the transverse anisotropy, which in most cases of oriented polymeric materials plays no role. From the 29Si experiments mentioned above, because of the axialsymmetry of the chemical-shift tensor we can obtain only an one-dimensional orientation information. A second angle needed for the analysis can be obtained by using the dipolar 29Si-29Si interaction.
Analogous investigation of carbon-based main chains is complicated by the existence of a large number of chemically different carbons leading to a lot of different lines in the spectrum. For resolving those from spinning sidebands, the two-dimensional PASS experiment was applied on polyester samples allowing us to distinguish between perpendicular and parallel orientations.
During the reorientation of a liquid-crystalline polymer in an switched external field, also the main chains have to be oriented into the new field direction. For some polysiloxanes the main-chain reorientation rate could be measured. It could be shown, that the reorientation of the mesogenic director is slowed down to that one of the main-chain segments.