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Originally published In Press as doi:10.1074/jbc.M212854200 on July 21, 2003

J. Biol. Chem., Vol. 278, Issue 42, 41189-41197, October 17, 2003
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Raman Microscopy and X-ray Diffraction, a Combined Study of Fibrillin-rich Microfibrillar Elasticity*

J. Louise Haston {ddagger} §, Søren B. Engelsen ¶, Manfred Roessle ||, John Clarkson **, Ewan W. Blanch {ddagger}{ddagger}, Clair Baldock §§, Cay M. Kielty §§ and Timothy J. Wess {ddagger}

From the {ddagger}Centre for Extracellular Matrix Biology, Department of Biological Sciences, University of Stirling, Stirling FK9 4LA, United Kingdom, Food Technology Centre for Advanced Food Studies, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark, ||European Synchrotron Radiation Facility, B.P. 220, F-38043 Grenoble Cedex, France, the **Department of Chemistry, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom, the {ddagger}{ddagger}Department of Biomolecular Sciences UMIST, P. O. Box 88, Manchester M60 1QD, and §§Wellcome Trust Centre for Cell-Matrix Research, Schools of Biological Sciences and Medicine, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom

Received for publication, December 17, 2002 , and in revised form, June 12, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fibrillin-rich microfibrils are essential elastic structures contained within the extracellular matrix of a wide variety of connective tissues. Microfibrils are characterized as beaded filamentous structures with a variable axial periodicity (average 56 nm in the untensioned state); however, the basis of their elasticity remains unknown. This study used a combination of small angle x-ray scattering and Raman microscopy to investigate further the packing of microfibrils within the intact tissue and to determine the role of molecular reorganization in the elasticity of these microfibrils. The application of relatively small strains produced no overall change in either molecular or macromolecular microfibrillar structure. In contrast, the application of larger tissue extensions (up to 150%) resulted in a markedly different structure, as observed by both Raman microscopy and small angle x-ray scattering. These changes occurred at different levels of architecture and are interpreted as ranging from alterations in peptide bond conformation to domain rearrangement. This study demonstrates the importance of molecular elasticity in the mechanical properties of fibrillin-rich microfibrils in the intact tissue.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Microfibrils are essential structural components of the extracellular matrix and are widely distributed in both vertebrate and invertebrate tissue, where they impart elastic properties on all dynamic connective tissues (1–7). Fibrillin-rich microfibrils have been examined extensively by both x-ray diffraction and electron microscopy techniques. Electron microscopy has revealed that microfibrils are macromolecular structures with a regular beaded appearance and a diameter of 10–14 nm. The average periodicity of these beaded microfibrils when isolated and in the untensioned state is ~56 nm, although a range of periodicities (33–165 nm) has been observed (4, 6). A fundamental axial periodicity of 56 nm was also observed by small angle x-ray scattering techniques, which revealed dominant 3rd and 6th orders. This is consistent with the presence of a regular array of microfibrils with a relative stagger of 56/3 nm and indicates a higher level of order in microfibrillar assembly (8–10).

A number of multidisciplinary studies have been conducted that investigated the structure-function relationship of fibrillin-rich microfibrils (4, 6, 8–11). Despite extensive investigation, however, the precise molecular arrangement (and consequently the basis of elasticity) of these microfibrils remains unknown. In the model described by Baldock et al. (4), a series of molecular folding events are proposed to occur during the assembly of fibrillin-rich microfibrils. These folds are believed to provide a basis for both the appearance and elasticity of microfibrils. This model is discussed in more detail below. This study utilized the techniques of small angle x-ray scattering (SAXS)1 combined with parallel Raman microspectroscopy to monitor changes in both the supramolecular and molecular structure of fibrillin-rich microfibrils (contained within the zonular filaments of ovine eye) under a variety of different conditions. The aim of this combined approach was to obtain greater insight into the basis of fibrillin elasticity.

Small angle x-ray scattering was utilized to characterize any changes in the supramolecular architecture of fibrillin-rich microfibrils following tissue extension. Any alteration in the periodicity of arrays of laterally aligned microfibrils can be easily monitored in this way. SAXS is a particularly useful technique as it allows the analysis of intact tissue samples in the fully hydrated state. Previous studies (10) have shown that the application of relatively low stresses produced only minor differences in periodicity. In contrast, further extension of the tissue resulted in a higher periodicity. This study aimed to characterize this phenomenon further by monitoring structural changes that occur during extension up to the limits of tissue extensibility. Previous work in this area was limited somewhat by the power of x-ray source available and by a low spatial resolution. In this study, the high brilliance beamline ID02 at the third generation synchrotron European Synchrotron Radiation Facility was utilized, which combines a highly collimated and parallel x-ray beam with a high resolution detector and low signal to noise level. The improved resolution and the intensity of X-rays available at this state-of-the-art beamline facilitated the investigation of fibrillin-rich microfibrils, both by enabling the detection of structures with a higher periodicity than previously seen and by allowing a vastly improved sample throughput.

In addition to this small angle x-ray scattering work was an investigation into possible parallel changes in molecular configuration. Raman spectroscopy is widely used in the study of protein structure and measures the inelastic scattering of light dependent upon internal molecular vibrations (12). Particular molecular groupings give rise to specific frequency shifts, generating characteristic spectra that provide information on protein conformation. Raman spectroscopy can be used to monitor protein secondary structure, domain-domain configurations, and the local environment of interacting side chains. This technique allowed examination of any changes in molecular conformation that arose upon the application of stress to the tissue.

Furthermore, any specific change in molecular configuration (induced for example by temperature changes, covalent modification or intermolecular interaction) can be detected by shifts in Raman band position. Raman spectroscopy is particularly useful for the analysis of aqueous systems as water generates very weak Raman scattering, thus producing little interference with the macromolecular signal. Raman spectroscopy was thus seen as an ideal method for investigating the role of molecular conformation on the elasticity of arrays of fibrillin-rich microfibrils in excised tissue.

The main molecular constituent of fibrillin-rich microfibrils is the glycoprotein fibrillin, although other molecules are also known to be associated (including microfibril-associated glycoprotein 1 and 2, latent transforming binding proteins and chondroitin sulfate proteoglycans) (13). It was anticipated that any alteration in protein conformation upon extension of these zonular filaments would produce an alteration in Raman spectra. These changes may indicate the levels of architecture that determine the elastic response.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Sample Preparation—Zonular filaments were dissected from ovine eye tissue as described previously for bovine tissue (8). Briefly, eyes were obtained within 24 h post-mortem from a local abattoir. Dissection of the posterior chamber of the eye produced an intact preparation of ciliary body, lens, and vitreous humor. Zonular filaments within this preparation were mounted securely on small aluminum frames (~1 x 0.5 cm, with a window of ~0.7 x 0.3 cm) using cyanoacrylate. Samples were kept hydrated using Tris-buffered saline (pH 7.2). Previous work was conducted using either phosphate-buffered saline (8, 10) or Tris-buffered saline (9). No difference has been found in sample behavior using either of these two buffers.

Small Angle X-ray Scattering—Small angle x-ray scattering of ovine zonular filaments was carried out on ID02 at the European Synchrotron Radiation Facility, Grenoble, France, using a 10 m sample to detector distance. Images were collected over 0.5 s on a Thomson x-ray Intensifier (TH 49–427) lens coupled to a FReLoN CCD camera (2048 x 2048 pixels). This detector has an active area of size 180 mm and a frame rate of 14 images (1024 x 1024 pixels) per second with a 14 bit nominal dynamic range. The wavelength of X-rays used was 0.09958 nm and the beam size at the sample was 300 x 300 µm. Three samples were analyzed and all exhibited similar properties. Images were obtained following a variety of different tissue extensions, which were estimated from the overall extension of the macroscopic tissue sample as described by Wess et al. (10). Briefly, the ends of each tissue sample were fixed and then separated by a distance proportional to the original rest length. Extensions were at ~0, 50 (data not shown), 100 (data not shown), and 150%. Images were also obtained following tissue relaxation back to the original rest length. These particular levels were chosen in order to analyze changes in microfibrillar structure that arise at the limits of tissue extension, immediately prior to tissue failure. Each sample was preconditioned before analysis to ensure that all had stable base-line mechanical properties. The level of pre-strain within each sample was estimated from the extent of arcing of the meridional and equatorial Bragg reflections. The rest length in this study was defined as the length at which the microfibrils could be seen to align and hence are taking up the macroscopic strain applied to the sample. Tissue extensions were determined subsequent to the measurement of this rest length. Samples were maintained in a hydrated state using Tris-buffered saline (pH 7.2) and analyzed between thin mica sheets. No beam damage was observed during these very short exposure times.

X-ray Data Analysis—X-ray diffraction two-dimensional images were calibrated using rat tail tendon collagen (which indexes on a 1/67 nm–1 meridional periodicity) and analyzed using in-house software that has been used previously to interpret type I collagen diffraction data. An empty cell background has been subtracted from each image. Typical diffraction images can be seen in Fig. 1. Integration of meridional intensities was carried out using FIT2D (14).



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  		FIG. 1.
X-ray diffraction images of ovine fibrillin-rich zonular filaments before, during, and after tissue extension. The images are of the tissue in the native (A); 150% extended (B), and relaxed states (C). The zonular filaments were aligned vertically in the beam. From the beam center the images extend to an R and a Z value of 0.0867 nm–1 (R and Z are, respectively, the radial and axial components of the central section of the cylindrical transform). Data were recorded on beamline ID02 at the European Synchrotron Radiation Facility (camera length of 10 m). An empty cell background has been removed from each image. Strong 1st and 3rd orders indexing on a fundamental axial periodicity of 55.4 nm are evident in A. B, following extension to 150%, the 3rd order (of the 55.4-nm lattice) is not visible, and the 1st order has moved to a lower angle indicating an increased fundamental axial periodicity. Tissue extension also led to an improved orientation of the microfibrillar bundles (as indicated by a decreased arcing of the Bragg reflections). Following relaxation of the tissue back to the rest length (C), both the 1st and 3rd orders reappear and return to their original positions, indicating that the structural changes induced by the applied strain are at least in part reversible.

 

Raman Microscopy—Raman microscopy measurements of ovine zonular filaments were performed on a Labram Infinity dual-laser spectrograph (Jobin-Yvon, Horiba, Lille, France) equipped with a Peltier cooled CCD detector. The measurements were carried out using a 532-nm frequency doubled Nd:YAG laser (100 milliwatts), a x100 objective, and a 600 g/mm grating resulting in a spectral resolution of ~13 cm–1. The spectra of a spot size of ~4 µm in diameter were acquired using an integration time of 60 s. Tissue extensions were performed as described for small angle x-ray scattering. Spectra of tissue in the native state were collected, following extension to 150% and after relaxation to the rest length. Spectra are shown in Figs. 2 and 3. The spectra of intensities were normalized to make direct comparison easier. The quality of the data and the low level of background noise made background correction unnecessary.



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  		FIG. 2.
Raman spectra of ovine zonular filaments before, during, and after tissue extension in samples A–C. Spectra were obtained using Raman microscopy with a frequency doubled Nd: YAG laser at 532 nm. The spectra of the fibrillin-rich microfibrils in the native tissue (i) and in the relaxed tissue (iii) are remarkably similar. In contrast the spectra of the zonular filaments following tissue extension to 150% (ii) showed a number of differences. This indicates that any differences in protein conformation that arise following tissue extension are largely recoverable upon relaxation.

 


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  		FIG. 3.
Difference spectra of Raman results (native and extended for each sample). A number of peaks and troughs are evident and common to all three difference spectra (A–C), corresponding to structural differences between the extended and native tissue. These features have been labeled with the appropriate wave number. Positive features are indicated by numbers at the top of the spectra, negative features are indicated by numbers at the bottom. Tentative assignments are given in Table I.

 


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  		TABLE I
Provisional assignment of Raman peaks in Figs. 2 and 3

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Small Angle X-ray Scattering—Representative images obtained from ovine zonular filaments in the native state, following tissue extension to ~150% of the rest length, and upon relaxation are shown in Fig. 1. The diffraction patterns obtained from the native tissue (see Fig. 1A) correspond to those reported previously for bovine eye tissue and exhibit a 1st order accompanied by a prominent 3rd order in the meridional series (8–10). Following tissue extension to ~150%, however, a significantly different image was produced (see Fig. 1B). In this case the meridional series was truncated and the positioning of the 1st order moved to a smaller angle. Differences were also produced in the equatorial series, with the extended tissue exhibiting an increased sharpness and indexing on a higher angle. There was also a decrease in arcing of both the meridional and equatorial peaks upon extension. Following relaxation of the tissue back to the rest length, however, there was an immediate return to the characteristic, albeit weaker, diffraction pattern, with first and 3rd orders visible (see Fig. 1C). This indicates that the extensibility of the tissue was reversible to this limit. Extension to higher levels resulted in tissue failure. Each of these findings and their implications are discussed in more detail below.

Raman Microscopy—Raman microscopy was performed on ovine zonular filaments in the native, extended, and relaxed states. The spectra obtained were highly reproducible from sample to sample, and representative spectra are shown in Fig. 2. The major peaks have been labeled with the appropriate wave number, and the structural features to which they correspond are detailed in Tables I and II (12, 15–18). The spectra of the native and relaxed tissue showed a high degree of homology to each other. This implies that changes in protein conformation, which are induced by tissue extension, are widely reversible upon relaxation. This also correlates with the findings from small angle x-ray scattering, which demonstrated a similar supramolecular arrangement within fibrillin-rich microfibrils in the native and the relaxed states.


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  		TABLE II
Locations of the amide I and III regions

 

Spectra produced by zonular filaments extended to 150% of the rest length, however, were significantly different from those derived from either the native or the relaxed tissue (see Fig. 2). Accurate identification of spectral changes that arose upon tissue extension was achieved by the generation of difference spectra. These were produced by subtracting the intensities of the extended samples from the intensities of the native samples. Peaks and troughs on the difference spectra correspond to regions where spectral changes have occurred and also allow visualization of the magnitude of these changes. These difference spectra are shown in Fig. 3, and the peaks of interest have again been labeled.

A number of alterations in Raman spectra occur upon extension of ovine zonular filaments. Changes occur at wave numbers 815, 831, 855, 921, 939, 1004, 1032, 1085, 1128, 1246, 1298, 1319, 1390, 1440, 1580, 1607, 1638, 1654, and 1663 cm–1 (see Figs. 2 and 3 and Tables I and II). Of particular note are differences in the amide I and III regions (which reflect protein secondary structure) and in the aromatic and aliphatic regions (which provide information on protein main chain structure and domain environments). Each of the changes in Raman spectra and their relationship to the model of molecular packing within fibrillin-rich microfibrils will now be considered in turn.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Small Angle X-ray Scattering—Previous studies (8–10) into the small angle x-ray scattering of zonular filaments were conducted using bovine ocular tissue. Legislation within the UK restricting the availability of bovine central nervous tissue has, however, more recently been extended to also include ocular tissue. Therefore, this study was carried out using ovine tissue as an alternative source of mammalian zonular filaments. The x-ray diffraction patterns obtained were remarkably similar to those observed using zonular filaments extracted from bovine eye. Three samples were analyzed, and representative images are shown here.

The images obtained from the native tissue (Fig. 1A) displayed a series of meridional Bragg peaks, which index on an average fundamental axial periodicity of 55.4 nm. The patterns exhibited a strong 3rd order at 18.5 nm, which correlates with previous findings (8–10) and suggests the staggering of adjacent microfibrils at a periodicity of one-third of the axial unit cell length.

Following tissue extension of 50%, no significant difference was observed in the three-dimensional organization of the microfibrils, as observed from the diffraction pattern (data not shown). This also corresponds to previous work that documented only minor changes in the axial periodicity of fibrillin-rich microfibrils upon tissue extension of up to 40 or 50% of the rest length (10). In contrast, the application of greater strains produced markedly different diffraction patterns. Tissue extension to 150% led to a truncation of the meridional series, with the 3rd order no longer visible (see Fig. 1B). The loss of a discernible 3rd order suggests that the existing supramolecular third staggered array of fibrillin-rich microfibrils has been disrupted in the 150% extended state. Extension also produced an increase in the fundamental axial periodicity of the microfibrils as indicated by a shift in the positioning of the 1st order (corresponding to 103.6 nm). This corresponds to a change of 87% in axial periodicity for a tissue extension of 150% and represents a non-linear extension of macroscopic and nanoscopic features of fibrillin-rich microfibrils. This finding was also similar to that reported by Wess et al. (10), where extension to 100% resulted in a shift to an 80-nm periodicity. In this study, tissue extension to around 100% resulted in a periodicity of ~82.5 nm (data not shown).

Equatorial reflections in the extended tissue exhibited an increased sharpness and extended to a higher angle (see Fig. 1B), indicating a decreased lateral spacing between individual microfibrils under applied strain. Furthermore, both the meridional and equatorial peaks became less arced upon extension. This indicates that the microfibrils within the zonular tissue are becoming increasingly aligned along the axis of the applied force.

Relaxation of the tissue back to the rest length re-established weak reflections in the meridional series, similar to those in the native tissue. A 1st order is visible above the background, indexing on 53.9 nm. A 3rd order is also apparent, occurring at 18.2 nm (see Fig. 1C). The recoverable nature of the x-ray diffraction pattern indicates that the characteristic 3rd staggered array of fibrillin-rich microfibrils can be re-established. This indicates that any supramolecular disruption that arises upon tissue extension is reversible and the tissue is elastic within the range studied.

These findings correspond with previous reports on the reversibility of the structural changes that arise following extension of fibrillin-rich tissues (8–11). In the findings reported by Wess et al. (10), it was reported that the characteristic 56-nm periodicity could be re-established following relaxation of tissues extended to 100%. The work presented in this current study demonstrates that following extension to even larger levels (150%), the original pattern can also be recovered. This provides further support to the role of this tissue reorganization in microfibrillar elasticity.

Furthermore, studies performed on isolated microfibrils reported similar limits of extensibility to those found here (4, 5). Baldock et al. (4) observed two major stable populations of microfibrils within samples extracted from tissue; the first had periodicities lower than 70 nm, whereas the second had periodicities above 140 nm. Similar results were reported in Eriksen et al. (5), who also observed two major stable populations of microfibrils within samples extracted from tissue. The first population had a periodicity of ~60 nm, whereas the second population was found in "highly stretched material" and had a periodicity of 140–150 nm. It was concluded from these studies that microfibrils were reversibly extensible in the range of ~56 to 100 nm and that irreversible deformation occurred at higher periodicities (i.e. in the microfibrils of 140–150-nm periodicity) (4, 5).

Both of these studies involved microfibrils that had been isolated from tissue, in contrast to the current study that examines the intact tissue. The findings reported here describe a reversible elasticity within the intact tissue of up to ~100 nm. Isolated microfibrils of these dimensions are reported by Baldock et al. (4) as being reversibly extensible, and as yet, microfibrils of longer periodicity (for example 160 nm) have not been detected in the intact tissue. In conclusion, microfibrils found within the zonular filaments of mammalian eye tissue are found to be reversibly extensible up to at least 103.6 nm of periodicity. This is in accordance with the literature which reports reversible elasticity in both intact and isolated microfibrils at periodicities below 140–160 nm, at which point the deformation becomes permanent (4, 5, 10, 11).

Raman Microscopy—Raman microscopy provides information on both long range and local features of protein structure. Consequently, information can be derived about any changes that occur in both secondary structure and in specific residues. The major alterations that arise upon extension of ovine zonular filaments are in the amide I and III regions and in the aromatic and aliphatic regions (in particular 1410 to 1470 cm–1) (see Figs. 2 and 3 and Tables I and II).

Correct interpretation of these data requires consideration of the molecular composition of fibrillin-rich microfibrils. Although a number of molecules are known to be contained within these microfibrils, the dominant component is the molecule fibrillin. Three isoforms of the fibrillin molecule exist, fibrillin-1, fibrillin-2, and fibrillin-3, which have a high degree of sequence homology. All are large glycoproteins (350 kDa) and possess multidomain structures. The individual molecules have an extended, rigid conformation and are ~160 nm in length.

The fibrillin molecule is dominated by 47 epidermal growth factor-like domains, 43 of which possess calcium-binding potential (cbEGF). These are interspersed by 7 transforming growth factor-{beta}-binding (TB) protein-like modules, each of which possesses 8 cysteine residues. Fibrillin-1 also contains a 58-amino acid proline-rich region near the N-terminal of the molecule. In fibrillin-2 this is replaced by a glycine-rich region; fibrillin-3 has a proline- and glycine-rich region. It has been proposed that this proline-rich region may serve as a "hinge region" in molecular folding (4). The molecule also possesses two-hybrid domains and unique N and C termini, which contain furin enzymatic cleavage sites, and are processed pericellularly as a prerequisite for assembly. Fibrillin-1–3 also contain multiple N-glycosylation sites (6).

EGF-like domains are widely distributed in nature and are found in more than 70 extracellular matrix proteins. These motifs are composed of between 40 and 50 amino acid residues and are characterized by a central {beta}-turn and a minor C-terminal {beta}-sheet. 6 cysteine residues interact to produce three disulfide bonds which stabilize the structure. A subset of these domains possess a characteristic amino acid pattern which confers calcium binding potential. Calcium has been shown to bind in an N-terminal pocket and is thought to increase the rigidity of the domain. A conserved aromatic residue is involved in stabilizing the binding site (19).

In contrast to the rigid cbEGF domains, the linkage region between the cbEGF domain and the sixth TB module in fibrillin-1 was found to be relatively flexible (20). Flexibility at TB-cbEGF linkages may therefore contribute to overall molecular folding and elasticity (4). The eight-cysteine TB modules fold into a globular structure composed of {alpha}-helices and {beta}-sheets. This structure is also stabilized by four internal disulfide bonds.

There are 13 tryptophans and 94 tyrosines in fibrillin-1, all are conserved between bovine and human sequences. One of the tryptophans is in the C-terminal domain and would be removed by furin processing. Of the remaining 12, 1 is in the N-terminal domain, 2 are found in cbEGF domains, and 9 are found in TB or hybrid domains. In TB and hybrid domains, 8 tryptophans are found at a conserved position between the 5th and 6th cysteines. This residue is thought to play an important structural role in the center of the hydrophobic core of this domain (20). The consensus sequence for the TB domains in fibrillin includes a tyrosine or phenylalanine at positions 8 and 64 and a tryptophan at position 45.

22 of the 94 tyrosines are found in TB or hybrid domains; 1 is in the signal peptide and 6 would be removed upon C-terminal furin processing. Two tyrosines are found in each of the N-terminal domains, C-terminal domains, and proline-rich regions; five are found in EGF domains, and the remaining 54 are found in cbEGF domains. These are predominantly found at two different conserved positions (42/54). The tyrosine at position 27 is involved in the calcium-binding site and is part of the calcium-binding motif. The tyrosine at position 35 has a key role in pairwise domain interactions; the side chain forms hydrophobic packing interactions with the glycine residue at position 25 in the following domain (see Fig. 4) (19). This interaction is important in stabilizing domains in their near-linear arrangement.



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  		FIG. 4.
Schematic representation of a cbEGF pair from fibrillin-1 (co-ordinates 1EMN [PDB] ) (19). Disulfide bonds are shown in yellow, and the residues involved in the calcium-binding site are colored green. Also shown is the conserved tyrosine residue at position 35 which has a role in pairwise domain interactions with a conserved glycine residue at position 25 in the following domain (both colored red).

 

The locations of the amide I (1640–1680 cm–1; carbonyl stretching mode) and amide III (1230–1310 cm–1; CN stretching mode) regions of the Raman spectrum in native, extended, and relaxed ovine zonular filaments are detailed in Table II. Significant differences are observed between the tissues, which correspond to different secondary structural features (12, 15). In particular, a major alteration occurs in the amide III region of the spectrum (Figs. 2 and 3 and Table II). The peak which occurs at 1246 cm–1 in the native samples exhibits a decreased intensity upon extension. This peak is re-established upon tissue relaxation. Also of note is the emergence of a relatively intense peak at 1298 cm–1 within the extended samples. This peak is absent in both the native and the relaxed states. Both of these peaks occur within the amide III region of the spectrum and therefore reflect secondary structural features of proteins. A peak at 1246 cm–1 is usually indicative of the presence of irregular or disordered chain structure, whereas a peak at 1298 cm–1 usually arises from {alpha}-helical structures (see Table II). The peak at 1298 cm–1 can also contain contributions from main chain CH deformations (12, 21).

Furthermore, these differences are also mirrored in the amide I region (1640–1680 cm–1; see Figs. 2 and 3 and Table II). In this region the shift is less obvious; however, the maximum of the amide I peak in both the native and relaxed samples occurs at around 1663 cm–1, which is usually concurrent with the presence of irregular and disordered domains or {beta}-sheet-like structures. Conversely the extended sample exhibits a peak at 1654 cm–1 which generally indicates {alpha}-helical content (12). It is important to note, however, that the location of the water bending mode at ~1645 cm–1 can sometimes obscure the amide I region and thus make interpretation more difficult (22).

It is therefore apparent that extension of fibrillin-rich microfibrils within zonular filaments induces a conformational change, with a decreased occurrence of irregular folds and turns and an increased {alpha}-helical content. As discussed in more detail below, microfibrillar extension may serve to unravel regions of the molecules, leading to an altered overall secondary structure. These changes are reversible upon tissue relaxation.

A major change is also observed in Raman spectra between 1410 and 1470 cm–1 (see Figs. 2 and 3 and Table I). This region of the spectra is largely dominated by CH and CH2 deformations, although aromatic residues also contribute. In both the native and the relaxed tissue the main peak occurs at around 1452–1465 cm–1, with a shoulder observed at 1428 cm–1. In the extended tissue, however, the peak at 1428 cm–1 disappears and the main peak shifts to a lower wave number (between 1440 and 1452 cm–1). A similar change was observed in the Raman spectra of the polymer isotactic polypropylene in the conversion from the solid (and partially crystalline) state to the melt (23). Solid isotactic polypropylene shows a main peak at 1458 cm–1 with a shoulder at 1435 cm–1. Conversely the melt exhibits a single peak at 1435 cm–1. Both peaks were in that case attributed to aliphatic deformations. The loss of the higher wave number peak was suggested to correlate with a change in chain conformation that occurs upon melting. The change observed in this study is also likely to arise through the alteration in protein conformation induced by tissue extension. The reported change in the amide I and III regions of the spectra indicates a conversion from random coil to {alpha}-helices in the extended state (see above). This conformational change will have obvious implications for long range chain ordering and inter-chain interactions, as reflected in the altering aliphatic deformations of these fibrillin-rich microfibrils. These changes appear to be reversible on relaxation of the tissue, as indicated by the similarity of the relaxed and the native spectra.

Changes in intensity are also observed in a number of other peaks, as detailed in Table I. A reversible change is observed upon extension of the zonular filaments to 150% at both 855 and 831 cm–1 (see Figs. 2 and 3 and Table I). The presence of a peak at ~850 cm–1 accompanied by a partner at 830 cm–1 is usually assigned to a tyrosine Fermi doublet. The ratio of the intensities at each of these two wave numbers is commonly utilized to indicate the degree of hydrogen bonding of the tyrosine residues. For example, a low value of I850/I830 indicates that the –OH group is acting as a strong hydrogen bond acceptor, whereas a high value generally correlates with donor status (12). In a study of the coat protein (pVIII) of the filamentous bacterial virus Ff, the absence of a peak at 830 cm–1 was attributed to a highly unusual or hydrophobic tyrosine environment (16).

In the results shown here the peak at 831 cm–1 is very weak in both the native and the relaxed spectra. The intensity increases slightly upon extension of the samples (see Figs. 2 and 3). In contrast, the relatively intense peak at 855 cm–1 exhibits a decreased intensity upon extension. It is hypothesized that this change in relative intensity is therefore caused by large changes in the hydrogen bonding status of the tyrosine residues within the fibrillin molecules upon extension of the tissue. More than 50% of the tyrosine residues within fibrillin-1 are found in one of two conserved sites within the abundant calcium-binding EGF domains. These tyrosines are involved in the calcium binding consensus sequence and in interactions between adjacent domains. This observed change in tyrosine environment could therefore reflect a change in the environment of the abundant cbEGF-like motifs.

The peak at 1343 cm–1 contains information on a number of different structural features, including the environment of tryptophan residues. Again this peak usually forms a doublet, with another peak at ~1360 cm–1. The ratio of the two intensities (I1360/I1340) provides information on the hydrophilic environment of the tryptophan residues, with a high value indicating hydrophobicity (12). In this case there is no obvious peak at 1360 cm–1, suggesting hydrophilicity. No change is observed in these peaks upon tissue extension implying that the environment of the tryptophan residues responsible for this region of the spectrum is not greatly affected by this tissue extension. Approximately 75% of the tryptophans within the fibrillin-1 molecule play an important structural role within conserved sites of the TB domains, close to the {alpha}-helical regions (20). These residues are located within the core of the domain, and it is possible that this region of the molecule is relatively resistant to any mechanical action. It is important to note, however, that changes are observed in the Raman spectra of a number of other peaks that contain tryptophan contributions (see Table I). It is therefore possible that the environment of at least some of these residues changes following tissue extension. Furthermore, a number of other structural features can also contribute to the peak at 1343 cm–1, including main chain deformations, meaning that definitive changes in tryptophan hydrophilicity may not be readily apparent from this particular part of the spectra (17).

A greater intensity in the native tissue as compared with the extended tissue is also observed at 939 and 1390 cm–1. These peaks typically provide information on protein side chain environments (both non-aromatic and aromatic), as well as protein main chain conformation (16, 18). The peaks present at 1004, 1085, 1128, 1580, and 1607 cm–1 display a greater intensity in the extended state. These again represent protein side chain and main chain vibrations (12), and the differences in these peaks upon extension and relaxation of these fibrillin-rich microfibrils may reflect a changing molecular environment within the tissue. These differences also appear to be reversible, due to the remarkable similarity between the native and relaxed spectra. The peaks at 815, 921, and 1638 cm–1 exhibit a fall in intensity upon tissue extension. As yet, however, these peaks remain unassigned.

Therefore, it is apparent that a number of reversible alterations occur in the Raman spectra of fibrillin-rich microfibrils following tissue extension. In particular, mechanical extension induces a conformational change with an apparent decrease in randomly coiled regions of protein and a relative increase in {alpha}-helical regions. These changes in secondary structure also produce alterations in the environment of specific residues within the microfibrils, as is evident from the observed differences in the aromatic and aliphatic regions of the spectra. These include changes in the hydrogen bonding status of a number of residues, in addition to differences in main chain interactions. Upon relaxation it appears that the protein spontaneously refolds to the original state, indicated by a return to the native spectrum.

Implications for Fibrillin Elasticity—A number of studies (6, 8–11) have demonstrated the reversible elasticity of these microfibrils, although the mechanism of this elasticity is unknown. The precise molecular alignment within fibrillin-rich microfibrils remains uncertain, although several models have been proposed (19, 24–26). The model proposed by Baldock et al. (4) describes a number of folding events which it is hypothesized contribute not only to the "beads-on-a-string" appearance of these microfibrils but also to their elasticity. This model is summarized in Fig. 5 and is described briefly below. For simplicity, only one row of aligned molecules is discussed; however, in microfibrils there may be 6–8 aligned rows of fibrillin molecules in cross-section.



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  		FIG. 5.
A model of fibrillin alignment in microfibrils (adapted from Baldock et al. (4)). Schematic diagram depicting a possible folding arrangement of fibrillin molecules in a beaded microfibril. A shows the structure of the fibrillin-1 molecule. Two N- and C-terminally processed molecules associate head-to-tail to give ~160-nm periodicity (B). Subsequent molecular folding events could generate ~100-nm periodicity and then ~56-nm periodicity (C and D). Fold sites predicted to generate periodicity of ~100 nm are at the N- and C-terminal junction and the proline-rich region. Fold sites predicted to generate ~56-nm periodicity are not known but may be at two or more TB-cbEGF junctions within the central region of the molecule, including TB3 (which has the longest linker sequence).

 

In this hypothesis, fibrillin molecules are thought to align in a parallel head to tail fashion. A series of folding events occurs that results ultimately in the generation of a stabilized untensioned polymer with a 56-nm periodicity. The inter- and intramolecular stabilizing forces within this structure are believed to account for the elasticity of the microfibril.

In the model, following secretion from the cell, fibrillin molecules are believed to associate through their N and C termini to produce a linear structure with a periodicity relating to the molecular length (~160 nm) (see Fig. 5A). Subsequent folding is thought to occur within the proline-rich region of the molecule, generating a "hinged" structure with a new periodicity of ~100 nm (see Fig. 5, B and C). This process allows alignment of exons 12–15 and 50–64, which aligns known transglutaminase cross-link sequences (4).

Subsequent folding event(s) are predicted to generate a structure of the characteristic 56-nm periodicity. The linkage regions between the TB and cbEGF domains are relatively flexible and are thought to contribute to this molecular folding. In particular, the so-called TB3 region (which contains a long linker region of 19 residues and is located before the central region of 12 cbEGF domains) is thought to be especially flexible. From experimental data it is proposed that folding arises within this region of the molecule, as illustrated in Fig. 5D (4). Several other such linkages within the central and C-terminal regions of the molecule may also become folded.

Based on observational studies, it was concluded that individual isolated microfibrils were reversibly extensible between periodicities of 56 and ~100 nm and irreversibly deformed at higher periodicities (up to ~160 nm) (4). Due to the correlation of these periodicities with the above model for molecular packing, it was hypothesized that reversible elasticity arises through the unfolding and refolding of the TB3-cbEGF region, producing a new periodicity of between 56 and 100 nm. Further extension of the microfibrils is believed to lead to unraveling of the proline-hinge region and disruption of the transglutaminase cross-links. It was therefore proposed that this process corresponds to an irreversible change in microfibrillar structure.

This model could be seen to correlate with the observations made using small angle x-ray scattering in this study. Minor tissue extensions (i.e. up to 50% of the original rest length) produced no significant differences in the axial periodicity of the microfibrils. In contrast, application of greater strains (up to 150%) results in the generation of a pattern with a longer periodicity (103.6 nm). Furthermore, a strong 3rd order is no longer apparent, implying a disruption to the proposed higher order staggered array of adjacent microfibrils thought to dominate the structure. The characteristic 56-nm periodicity was in each case recoverable following tissue relaxation. Extension to higher levels resulted in tissue failure. These observations describe macromolecular changes to physiological bundles of fibrillin-rich microfibrils upon application of strain to the system. It is possible that these effects correlate with those observed with isolated microfibrils in vitro (4), with reversible extension up to ~100 nm and the proposed unfolding of the TB-cbEGF domain (see Fig. 5C).

In parallel with the observations made by small angle x-ray scattering, the Raman microscopy of zonular filaments described here represents the first attempt to correlate the elasticity of intact tissues containing fibrillin-rich microfibrils with changes in molecular conformation and domain-domain interactions. Samples examined by both techniques were subjected to identical experimental conditions to allow direct comparison of the data. The observations made by Raman microscopy could also be seen to correspond to the hypothesis outlined above. A reversible alteration in Raman spectra was observed following tissue extension to 150%. If the elasticity of the tissue arises at least in part from molecular unfolding and refolding, then these types of changes would be expected as differences arise in the conformation of the protein.

The native tissue had spectra characterized by a relatively low {alpha}-helical content, compared with a higher irregular and {beta}-structural content. In contrast, the extended sample exhibited a depleted irregular and {beta}-sheet containing profile and a higher {alpha}-helical content. The abundant calcium-binding EGF-like domains, found throughout the fibrillin molecule, are known to contain both a {beta}-sheet and a {beta}-turn (19). The TB domains, which are less prevalent, contain two {beta}-sheets and two {alpha}-helices (20). The observed changes in this study clearly demonstrate that a reversible change occurs in the secondary structure of fibrillin-rich microfibrils following tissue extension. It is hypothesized that this mechanical action leads to an unfolding of the molecules within the microfibrils, as proposed by Baldock et al. (4). The molecular reorganization induced by this process results in a change in Raman spectra. Microfibril extension appears to produce an unraveling of certain structural features (in this case the irregular and/or {beta}-turns and folds), and a relative increase in the proportion of other features, such as the {alpha}-helix, which are normally relatively low in prominence in the fibrillin molecule. Upon relaxation of the tissue, the protein appears to refold into the native configuration.

A change in molecular configuration upon tissue extension is also supported by differences in the aromatic and aliphatic regions of the Raman spectra. A large change is observed in the aliphatic CH/CH2 region of the spectrum between 1410 and 1470 cm–1. It is proposed that this reflects a change in protein main chain interactions induced by the alteration in protein conformation. A change in tyrosine environment following tissue extension is also observed. These residues are prominent in the cbEGF domains of fibrillin. It is proposed that any molecular rearrangement that occurs following microfibrillar extension exposes these domains to different forces and different parts of the molecule than in the relaxed state. The consequent change in interaction and environment produces a corresponding change in Raman spectrum. In both cases the original conformation appears to be largely recoverable upon tissue relaxation, as indicated by a return to the native spectrum.

In conclusion, this study represents the first attempt to interpret the elasticity of fibrillin-rich microfibrils on both a macromolecular and submolecular level. It is apparent changes occur in both the packing of the microfibrils and in the conformation of the protein constituents of these microfibrils under the application of large strains (up to 150% tissue extension). The changes that occur appear to be largely reversible upon relaxation. Further investigation of these structural phenomena is warranted to establish in greater detail the structural changes that occur and the uniformity of effect. Future work could include structural investigation of recombinant peptide regions of the fibrillin-1 molecule in an attempt to define more precisely the changes observed in this study. Time-resolved studies to investigate the rate of change of SAXS and Raman spectra using a controlled mechanical testing regime could also be performed in the future. This would reveal the possible existence of intermediate structures and establish the limits of elasticity, leading to an enhanced understanding of the functioning of fibrillin-rich microfibrils.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by Grant 98/S15326 from the Biotechnology and Biological Sciences Research Council. To whom correspondence should be addressed. Tel.: 44-1786-467814; Fax: 44-1786-464994; E-mail: j.l.haston{at}stir.ac.uk.

1 The abbreviations used are: SAXS, small angle x-ray scattering; cbEGF, calcium-binding epidermal growth factor; TB, transforming growth factor-{beta}-binding protein-like domain; EGF, epidermal growth factor. Back


   ACKNOWLEDGMENTS
 
The support of the Centre for Advanced Food Studies MRI in providing funding and access to the facilities at the Kgl. Veterinærog Landbohøjskole (Royal Veterinary and Agricultural University) is gratefully acknowledged as is the support of the European Synchrotron Radiation Facility.



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