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Peer-reviewed

Research Article

Variation in the Helical Structure of Native Collagen

• Joseph P. R. O. Orgel,
• Anton V. Persikov,
• Olga Antipova

10

• Published: February 24, 2014
• https://doi.org/ten.1371/periodical.pone.0089519

Figures

Abstract

The structure of collagen has been a thing of marvel, investigation, and argue for the better role of a century. There has been a peculiarly productive menstruation recently, during which much progress has been made in better describing all aspects of collagen construction. However, there remain some questions regarding its helical symmetry and its persistence within the triple-helix. Previous considerations of this symmetry have sometimes confused the motion picture by not fully recognizing that collagen structure is a highly circuitous and large hierarchical entity, and this affects and is effected by the super-coiled molecules that make it. Nevertheless, the symmetry question is not trite, but of some significance as it relates to extracellular matrix organization and cellular integration. The correlation betwixt helical construction in the context of the molecular packing system determines which parts of the amino acrid sequence of the collagen fibril are buried or accessible to the extracellular matrix or the cell. In this study, we concentrate primarily on the triple-helical structure of fibrillar collagens I and Two, the 2 most predominant types. By comparing Ten-ray diffraction data collected from type I and blazon Two containing tissues, we point to evidence for a range of triple-helical symmetries being extant in the molecules native environment. The possible significance of helical instability, local helix dissociation and molecular packing of the triple-helices is discussed in the context of collagen's supramolecular organization, all of which must bear upon the symmetry of the collagen triple-helix.

Citation: Orgel JPRO, Persikov AV, Antipova O (2014) Variation in the Helical Structure of Native Collagen. PLoS ONE ix(2): e89519. https://doi.org/x.1371/journal.pone.0089519

Editor: Collin Chiliad. Stultz, Massachusetts Institute of Technology, U.s. of America

Received: August 21, 2013; Accustomed: January 21, 2014; Published: February 24, 2014

Copyright: © 2014 Orgel et al. This is an open up-access article distributed nether the terms of the Artistic Commons Attribution License, which permits unrestricted utilise, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Use of the Avant-garde Photon Source was supported by the U.S. Section of Energy, Bones Energy Sciences, Part of Scientific discipline, under contract No. Due west-31-109-ENG-38. BioCAT is a National Institutes of Health-supported Research Centre (RR-08630). The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institutes of Wellness. This work was also supported by the National Science Foundation (Grant #MCB-0644015 CAREER) and this material is based upon work supported by, or in part by, the U.S. Army Research Laboratory and the U.S. Army Research Office under contract/grant number W911NF 09-1-0378. The funders had no role in study pattern, data drove and analysis, determination to publish, or training of the manuscript.

Competing interests: Delight note that Joseph Orgel (PhD) is a fellow member of the editorial board. This does not change the authors' adherence to PLOS ONE Editorial policies and criteria.

Introduction

The structure of fibrillar collagen is complex and multifaceted (Figure 1) [i], [two]. In general, most structural studies of collagen have focused on either its triple-helical limerick or its fibrillar arrangements. However, collagen'southward molecular packing is as well key in understanding its overall structure and function [one]–[3]. This packing structure is directly effected past and affects both collagen'southward helical and fibrillar arrangement [4], (literally) providing the framework to understand otherwise context-less stretches of peptide sequences that cells recognize and interact with [1], [2], [5], [6].

Figure 1. Greatly simplified organizational hierarchy of fibrillar collagen structure (from polypeptide to fibril)

A. The collagen-forming polypeptide chains comprise a big helix-forming domain with the repeat amino acid sequence Gly-X-Y, where X and Y are occupied by Pro or Hyp more frequently than other residues, merely merely business relationship for approximately 1/6 of the total amino acid content (see for instance homo sequence: ExPASy sequence data bank codes; P02452 and P08123). An arrow points to the figure element that shows that three polypeptides form the collagen monomer. The large triple-helix (super-helix) domain of approximately 300 nm in length is flanked by not-helical telopeptides (Northward and C, shown). The 6–8.6 nm dimension indicates the echo of the triple-helix (36; 37). B. Collagen molecules are staggered approximately 67 nm from one another in the formation of microfibril aggregates. The microfibrils are D-periodic (D = 67 nm), and in each D-period, 2 monomers coil, or partially coil, around each other giving the appearance of another helix-similar feature in the structural hierarchy (three). C. Cross-exclusive view of the collagen molecular packing of a blazon I collagen fibril (11). Each circumvolve represents one collagen molecule in cross-section (at the axial level of 0.44D). at the 0.44 D position. Side by side to B to C arrow, cross-section of an isolated microfibril. D) Archival image (Orgel laboratory) of the wide bending fiber diffraction blueprint of type I collagen from rat tail tendon. The distinctly dissimilar but superimposed non-crystalline and crystalline diffraction patterns are indicated. Previous fiber diffraction studies of collagen's helical construction take concentrated on the not-crystalline office of the pattern, in this present written report, nosotros analyze crystalline diffraction data.

https://doi.org/10.1371/journal.pone.0089519.g001

X-ray fiber diffraction has been a critical tool in elucidating the structure of the molecular packing arrangement, which in plow, has allowed insights into other aspects of the internal molecular arrangement [three]. The highly crystalline packing of collagen molecules in the direction of the collagen helix in some tissues in particular, allows the drove of crystalline diffraction patterns [7]. Different many fiber diffraction patterns which show but the unsampled molecular transform of the sample (non-crystalline diffraction arising from the helical symmetry), these patterns also contain Bragg peaks originating from the well ordered axial and lateral packing of collagen molecules into fibrils (Effigy 1D). This crystalline diffraction from collagen tissues may exist treated as analogous to that from a single macromolecular crystal. Except that it arises from many fibrils within the sample, giving rise to cylindrical and rotational averaging effects and the inherent complications that its multi-crystallite composition imposes (such as overlapping Bragg peaks and in-coherent scatter in the off-meridional regions) [four], [viii], [9]. The meridional section of the diffraction pattern does not suffer from these issues however. Furthermore, it extends to medium angle resolution and demonstrates a potentially very high degree of order within the axial packing arrangement which is in the same orientation as the helix [4], [10].

Such crystalline diffraction data, arising from the centric and lateral packing, has been used to decide the molecular packing construction of type I collagen in situ [four]. In this previous work, both the native and 2Fo-Fc electron density maps, constructed from experimentally adamant phases and observed amplitudes, showed expert agreement. The observed diffraction and the simulated pattern calculated from the model fitted to the experimental electron density map also showed skillful agreement. In an endeavour to develop the about accurate model possible from the available data, coordinates composed from high-resolution collagen-like peptide structural data [11] were fitted into the low-resolution electron density map, which is essentially a molecular envelope. This arroyo is analogous to that commonly used with cryo-electron microscopy or SAXS information whereby college-resolution structural data are "docked" into the molecular envelopes defined past the low-resolution data, [12], [13]. Every bit such, it would be inappropriate to expect the structural models from such studies to exactly friction match that derived from high-resolution single-crystal crystallography or multidimensional NMR of molecular model peptides. In the same vein of thought, however, since they are experimentally determined and not solely in silico models they are of significant value if used appropriately (and benefit from the same highly advanced force field calculations used in pure modeling studies [14], [15] while retaining explicit experimental data verification).

In the present report we consider what diffraction data arising from the molecular packing construction of collagen tin say regarding the helical organization of the fibrillar collagen's. Our objective is non to show that one particular helical symmetry is prevalent, but to demonstrate that there appears to be multiple conformations in fibrillar collagen'southward helical domain that include variations other then the ascendant symmetry question. This possibility has been predicted from brusque model peptides of collagen or collagen-similar sequences just has not yet been shown for the full molecule in its natural context [xvi], [17]. In connection to the symmetry of the helix, our arroyo avoids potential pitfalls in model plumbing equipment to 'less-than-perfect' helical diffraction by primarily using the Patterson function to detect helical periods in the crystalline meridional data of two different fibrillar collagen systems. Further observations are fabricated via the employ of experimentally determined models and 'platonic' helical models composed to fit expected helical symmetries described in the literature [11], [16], [17]. Finally, since the triple-helix of blazon I collagen deviates from the expected triple-helix structure in ways that are not just symmetry related [iii], [16], [18], [nineteen], we calculated sequence based predictions of local triple-helix stability to see if these features are only stability based or if other factors (such every bit lateral packing and bends in the molecules) might play a role in defining collagen's helical construction. If these variations, such every bit helix 'puckering', always correlate to depression thermal stability regions then it could be causeless that lateral packing and other molecular interactions with collagen accept little effect on its helical structure.

Using meridional information from type I and type Two collagen containing tissues we accept detected periodic functions in the native Patterson function that may indicate the underlying helical symmetries of both these fibrillar collagens. Together with structural information obtained from an earlier three dimensional packing structure of type I collagen [3], [4], this provides insight into variations in the helical organization of the collagen triple-helix inside the fibrillar collagen's 'helical domain'.

Materials and Methods

Fiber Diffraction and Coordinate Information

X-ray cobweb diffraction data from native, hydrated, rat tail tendon and lamprey notochord were obtained in previous studies [4], [10], [xx]–[22]. The scaled amplitudes of the central, meridional section of each data set were used to calculate Patterson functions, whilst the contributed coordinates from Orgel et al., 2006 [4] were used to for displaying aspects of collagen's molecular and packing construction (Figure 1).

The type II information is published in supplementary information of Antipova and Orgel [22], the type I information is to be found via the linked RCSB codes (3HR2 and/or 3HQV).

PDB Models

Structure factors were calculated for each model to generate model based construction factors for comparison with those adamant experimentally. These were used in the Patterson Part calculations (below).

The coordinates of an experimentally determined type I collagen model construction (RCSB ID: 3HR2) (3; 14) that contains the fibrillar conformation of the collagen molecule (including its packing) were used for comparison with native collagen.

The crystal construction of the (PPG)_x triple-helical peptide from crystals grown in a microgravity environment with an "ideal" 7/2 symmetry (RCSB ID: 1K6F [23]) was used to simulate an "ideal" full-length collagen with perfect 7/two helical twist. The third position prolines were substituted to the Hyp (4-R-hydroxyproline) residues and the construction was translated into full-collagen length with (Gly-Pro-Hyp)₃₃₈ sequence.

All available high-resolution crystal structures at the time this enquiry was conducted, were used to get as close as possible to the arcadian triple-helix with ten/iii symmetry [eleven] and the portion of type Three collagen peptide in detail (PDB ID: 3DMW [17]) was used as a design with an "ideal" 10/3 helical twist. This included residues 6–20 of chain A, residues 6–17 of chain B, and residues half dozen–17 of chain C of 3DMW, with 33 residues in total, making one triplet overlap for a complete menstruum. This structural unit was mutated into (Ala-Ala-Gly) repeating construction (with zero imino acid content to disfavor the seven/2 helix). To accommodate positions of all atoms, the idealized alpha-carbons were stock-still and the rest of the structure was energy minimized using AMBER forcefield [24]. Finally, this idealized construction was translated to give a full-length (helical portion) collagen molecule with (Ala-Ala-Gly)₃₃₈ sequence and the ideal 10/3 helical twist.

Each structure was superimposed (without changing its internal structure) on the native blazon I collagen molecule to give the same degree of relative tilt to the unit cell [9]. Ii more models were composed from the GAA and GPO coordinates by substituting the type I collagen sequence onto each structure coordinate atoms (GAA colseq and GPO colseq respectively). The side chains of the amino acid sequence of type I collagen (that seen in 3HR2) replaced the corresponding 10 and Y-positions, glycine positions were unaltered. The structure factors were calculated for each model then that Patterson functions could be fabricated.

Patterson Office

The i dimensional Patterson function for the 00l (meridional diffraction series) was calculated:

Where 00l are the Miller indices of the (1 dimensional) unit cell, |F_00l| the amplitude and west is a bespeak in unit of measurement prison cell space. The significance of the function is that the peaks in a Patterson function refer to the distances betwixt repeating electron dumbo regions (such every bit helically organized electron densities) within the crystalline unit of measurement cell of the diffraction sample. The serial terms are the scaled square amplitudes [iv], [10], [22]. In brusk, the Paterson function is a pair correlation part for electron density, the peaks of which reveal periodicities in interatomic distances.

Triple Helical Stability

The triple-helical stability was calculated via the "Collagen Stability Reckoner" [25]: http://compbio.cs.princeton.edu/csc/contour.html, for the rat sequence helical domain (uniprot P02454, so as to match the type I experimental data). The plot was inverted so that increasing instability gave peaks rather than troughs. This allowed the results to be compared straightforwardly with a plot of helix dissociation. The latter was calculated from the boilerplate Calpha deviation of the 'relaxed' model (3HR2) from the 'rigid' model (3HQV) of the in situ collagen helix, afterwards the method of Perumal et al [3].

Results and Discussion

Previous analysis of the model and electron density of the type I collagen in situ packing structure [four], [10], [26], [27] showed that the fibrillar collagen molecule is composed of triple-helical and non-helical domains (Figures 1 and 2). The lateral packing of neighboring collagen molecules is quasi-hexagonal, the intermolecular cross-links that help maintain this relationship being known to be formed via the non-helical telopeptides. The (GPO)_v domain direct proceeding the C-terminal telopeptide (Figure 2) is the collagen sequence about closely related to studies of collagen-similar peptides such as (GPO)₁₀ [28]. The electron density of this region indicates a well-formed triple-helix for most of this domain, except for the last 2 repeats where information technology connects to the non-helical telopeptide. Here the more than bulbous electron density expands beyond the normal diameter of the triple-helix as it describes the outline of the folded α1 chains. This indicates the triple-helix begins to associate at the (GPO)₅ region as suggested previously [29], [30], but must transition from the non-helical conformation to the triple-helical conformation over one or more GPO repeats. Other sections of the 'triple-helical domain' are similarly not entirely triple-helical. For instance, the α chains are more dissociated from the center of the triple-helix than data from high-resolution, model peptides, can detect due to their short length and required sequence bias (loftier imino acid content) [iii], Figure 3.

Figure 2. Helical and non-helical system of collagen.

The not-helical, folded C-last end of the collagen molecule (top) extending from the triple-helical region (below). The electron density of neighboring collagen molecules can be seen along side the chain traced segment (cherry). The GPO_five domain is indicated in white.

https://doi.org/ten.1371/journal.pone.0089519.g002

Figure 3. Thermal stability versus α chain triple-helical dissociation.

A) Thermal stability plot [25]. B) Comparison of local predicted stability variations with local helical dissociation. Blue lines mark a noteworthy correlation between peaks that signal thermal instability of the helix and where the helix is as well dissociated (see role C). Red lines bespeak noteworthy areas where in that location is not a correlation. The helix is calculated to be thermally stable but the low resolution structural data indicates the triple-helix to be relatively dissociated at room temperature. Or vice versa, the stability plot indicates a well formed helix while the structural data shows a relatively disassociated i. Some of the places were at that place is no correlation (stable helix but construction shows a dissociated ane) are located at points of molecular inflection (bends, meet Figure ane and 3 and electron density [4]). Thicker lines indicate more than significant discrepancy/correlation, unmarked areas are thought to testify more or less expected similarity between the two plots (A and C). C) dissociation of peptide bondage: deviation between the calculated relaxed model (via forcefulness field calculations against diffraction information) and the starting stringent model (from loftier resolution model peptide data) of the collagen triple-helix. Sequence numbering includes the N-telopeptide residues. This is an gauge of triple-helix dissociation. The magnitude of dissociation of the iii peptide chains are shown as a local average (black line), forth with the global boilerplate (blue line) and two times standard departure (ii*σ, light blue line). ane–6 and * indicate meaning bends in the collagen molecule as determined from the electron density of in situ fibrillar collagen data.

https://doi.org/ten.1371/journal.pone.0089519.g003

Helical instability versus helix dissociation

Is helix dissociation, a pregnant deviation of helix structure from the ideal (and therefore also perfect symmetries), a persistent structure or due to helical instability?

Several short regions of the fibrillar packing structures [four] helical domain are aptitude (and there is a pregnant change in direction of the molecular helix before and after the curve) and accept a larger diameter then the remainder of the density representing the triple-helices. The triple-helical models (3HR and 3HQV) fitted to these electron densities adapt these bends by bulging outward (a triple-helix local disassociation) presumably also to accommodate the electron-density which has a larger diameter then the boilerplate helix in these locations. In a subsequent investigation [3], it was institute that the α bondage are significantly disassociated at several other sites (Figure 3). The points at which the helix is most disassociated are at those points of molecular inflection (equally indicated, Figure 3). That is, where the molecular segments bend within each D-period to accommodate their re-organization every bit they progress into the adjacent D-flow (see microfibril in Figure i).

Local disassociation of the triple-helix appears to coincide with both regions of calculated relative instability and stability. In Figure 3, regions 1-6 correspond to the bends that suit molecular segment re-arrangement. Regions 2–iv show a correlation between relative peaks in thermal instability and triple-helix dissociation. Regions 1, v and 6 bear witness regions of relative helical stability with relatively large triple-helix dissociation. The latter case is consequent with the proposal that deviation from the helical ideal is not an artifact from thermal fluctuation, but a persistent structure (see ruddy lines, Figure iii). This assertion is supported too from the molecular packing structures electron density [four], or otherwise those thicker sections of electron density where the helix bends, would not be visible to X-ray diffraction. I.e, those bends must persist over millions of crystallites (fibrils) inside the tissue sample.

In full general, these and other areas of significant dissociation correlate well with the calculated local stability of the triple-helix (Figure 3A) albeit with some evident differences. Some areas that show helix dissociation where the thermal adding proposed an increased stability, and vice versa (not-dissociation in regions of proposed low stability) are indicated in Figure 3B with red lines. This latter point does not betoken a discord, but possibly shows that at to the lowest degree some of these dissociated areas are disassociated due to the bend of the helix just are otherwise stabilized by the closely spaced neighboring helices. This possibility is supported past the fact that such areas appear to cluster around the inflection points of the molecule (marked 1–6 and *, Figure 3). All the same, the majority of the disassociated triple-helical areas correlate well with the depression thermal stability regions, indicating that local amino acrid sequences by and large decide fluctuations in triple-helix stability and local helix dissociation.

When taken into account along-side the periodicities institute in the 10-ray diffraction terms from type I and Ii collagen showing intermediate structures to the ten/3 and 7/2 helices (see below), these information also betoken that collagen's helical structure is more than various than that observed in curt relatively elementary model peptides. This should not exist as well surprising equally there are factors not easily accounted for in most studies of collagen helix structure or stability: most of the triple-helix is closely packed next to neighboring triple-helices and at other times are closely associated with ECM ligand binding proteins at the fibril surface [six]. The potential helix stabilizing effect of inter-molecular packing may explain instances were the calculated stability indicates an unstable helix, while the structural data indicates the helix is well formed (red lines, Effigy 3 B). This is seen especially for the very low thermal stability region between 75 and 100 that with a small exemption, shows a well formed helix despite low predicted stability. Regions in Figure 3C marked 1, v and 6 too testify like discrepancies, merely all correspond to regions of helix bending that occur in a region of the D-flow (the gap region) that is heavily substantiated with the proteoglycan decorin [31], [32] that could contribute to stabilizing those corresponding regions of the triple-helix.

Variations in helical symmetry

In that location are currently two well described symmetries for the collagen triple-helix: the Rich and Crick/Fraser et al model describing the triple-helix and a x/3 helical symmetry for it [33], [34], and the Cohen and Bear/Okuyama et al., [28], [35], [36] 7/2 helix symmetry structure. The average conformation of both is a left-handed triple-helix with either 10 repeating GXY triplets in three turns (10/3) or 7 repeating GXY units in ii turns (vii/2). Each possess unique periodic values, a pitch of 2.86 or two nm for the 10/3 and seven/2 respectively, and a 'true repeat' [37] of 8.58 and half dozen nm for 10/3 and 7/2 respectively (Figure four). There has been a considerable amount of give-and-take regarding which of these structures represents 'real' collagen structure, with investigations of amino acid and imino acid rich brusk collagen-like peptides and re-analysis of their data [11], [38]–[41] to observe evidence of 10/iii or seven/2 helical twists [38]. Others re-investigated the molecular transform fiber diffraction data to examine the same question [34], [42]. Each of these studies has its inherent strengths and weaknesses. The collagen-like brusk peptide studies offer loftier-resolution structures and suggest that high imino acid content leads to helices that are closer to 7/two symmetry, while the peptides with low imino acid content and lower stability tend to accept a helical twist (but not necessarily the expected period repeats) closer to the 10/3 (26). The fiber diffraction studies of the molecular transform, have relied on discrete model based studies, which introduces the concern of non-unique solutions or that they may overlook that a possible range of conformations exist [42]. In addition, the fact that early on studies of the helical conformation relied on highly stretched samples ∼ten% [34] leads to the possibility that the triple-helix itself may too be stretched (in this case in favor of the 10/3 helix). Other studies may find alternate conformations from diffraction data nerveless nether weather condition that may not be the aforementioned. Fifty-fifty a cobweb diffraction written report that did not suffer from these model ambiguities and made use of taut but not highly stretched tendons [4] could non (directly) address the question of helical symmetry properly due to its anisotropic resolution. Still, the crystalline diffraction information from this [4], [10] and related studies [22] may be used to await for bear witness of helical periodicities associated with one helix class or another without a priori model bias.

Figure 4. Helix net map of the ten/3 (A) and 7/2 (B) triple-helix models.

The unit of measurement height of the α-peptide chain (h) and the superhelix (h_sh), the pitch and helix true repeat periods of each helical symmetry is as indicated.

https://doi.org/10.1371/journal.pone.0089519.g004

The native Patterson function plots of meridional information (relating to the axial, including helical, construction) from rat tail tendon (collagen type I) and lamprey notochord (collagen type 2) prove several common features (Effigy v). Of interest to this study is the range of periodicities detected between 0 and 11 nm (Figure 5B). Although the unit top of both the 10/3 and 7/2 helical models at ∼0.86 nm (Figures 5 and 6) is below the detectable threshold of the plots (the 'self' menstruation of the Patterson map is steep and uninterpretable for this region), periodicities above 1.5 nm for blazon I and 1.9 nm for blazon Two are clearly seen as peaks for both data sets (Figure 5). Since the type I dataset has a resolution of ∼0.5 nm and the type Ii 1.9 nm, periodicities detected above these respective values are within the probable competence of the data. Periodicities that may stand for to the helical pitch and true repeat are clearly seen (Figures v and 6) and are recorded in Table 1 (along with the other periodicities observed).

Figure five. Patterson functions of the type I and II collagen 00L (meridional) series.

A) Patterson function from 0.0–0.5D, the inverse (0.5–1.0) half of the Patterson function is not shown. The fractional distances between periodicities indicated in the functions has been multiplied past 67 nm (the length of the one dimensional unit cell – the D-menstruation) for comparison with the helix symmetry periods. B) Enhanced view of the Patterson office range of interest for the helix symmetry periodicities. C) Table of key helix periodicities for comparison with A and B (run across likewise Figure 4).

https://doi.org/x.1371/journal.pone.0089519.g005

Figure 6. Patterson functions of collagen model structure factors 00L (meridional) series.

A) Comparison of GPO (seven/2 model) and GPO with collagen sequence threaded to check if amino acid sequence effects periodicities detected by the Patterson function. It does non announced so. B) As (A) except for GAA (10/3 model). C) Patterson functions of collagen types I and II are compared with those from the GAA and GPO coordinate models with the collagen sequence threaded onto them. The semi-transparent arrows marking: ruby, the maximum of the GAA (10/iii) helix model pitch and echo periods, the black arrows mark the collagen I and II corresponding positions for these periods. Notation that the collagen experimental data show periods that are longer and so the 7/two and practice seem to almost achieve the 10/3 expected range. This could be interpreted to hateful that both helical symmetries are found in native fibrillar collagen in addition to other possible conformations.

https://doi.org/x.1371/periodical.pone.0089519.g006

A pitch with a true repeat ratio of 3∶i, a non-integral helix (an integral helix existence one were the true-repeat and pitch are the same, such as in B-form DNA), would bespeak that the pitch and repeat values are well paired (viii.58/2.86 = 6.0/2.0 = iii.0). The observed, average periodicity that appears to correspond to an average pitch value of 2.43 nm (from an admittedly wide range) provides a production of two.6, iii.18 or 4.06 when possible repeat values of 6.three, 7.7 and ix.85 nm are used (observed values in the Patterson functions). Both the 2.43 nm pitch and 7.7 nm repeat values being intermediate betwixt the x/3 and 7/2 helical catamenia values. Still, the being of three singled-out period values that may represent to helical repeats (the 6.three, seven.7 and nine.85 nm values) could indicate at least iii distinct helical symmetries in collagen samples with minimal stretch. The fact that the ii.43 nm 'pitch' periodicity is broad, could also betoken the existence of both the 10/3 2.86 nm and 7/2 2.0 nm pitches. As, Table one indicates, that the average of the 7.vii and 9.85 nm 'echo' periodicities is very close to the 'perfect' x/3 helix repeat whilst neither individual value is advisable. The same is true of the 7/ii helix repeat value, half-dozen.iii nm existence greater than that expected.

Given the stiff showing of periodicities that are in the range of the helical symmetry periods expected, it would be straightforward to envision regions of the collagen molecules that conform to the ideal 10/3 or vii/2 helical symmetry. The values seen in the Patterson maps (in Figures v and 6) stand for to averages of structures found inside the samples, even though they do appear to cluster effectually three observed echo values. The ideal 10/3 structure periodicity is not observed, but two values (7.seven and ix.85 nm) may correspond evidence for the existence of related symmetries whose average is 10/3. Neither is the ideal seven/2 structural periodicity observed, although the repeat value of 6.3 nm is closer to the expected ideal than its corresponding x/iii structure. A caveat to be considered hither, is that stretching for at to the lowest degree tendon samples does in fact represent something not unlike its native state. Information technology is quite possible that the work of Fraser et al [34] represents an accurate definition of 10/3 structure in tendon samples, if that stretching causes the normalization of the molecules helical symmetry towards the larger repeat values. That the type I and type 2 collagen samples produced similar, but not identical values may also indicate variation in the molecular conformation of the helix as per its tissue context. Information technology is besides a possible consequence of the blazon Ii collagen notochord samples being stretched, perhaps to a greater extant and so the blazon I tendon samples to aid in the recording of meridional diffraction over that of lateral packing diffraction from a second population of fibers [22]. This could also explain why the type Two Patterson functions give indications that are plainly closer to the ideal 10/3 parameters then that from the type I information used in this study [iv], [10], Figures five and 6.

Although Fraser et al [34] and Okuyama et al [42] when comparing a priori helix models against molecular transform fiber diffraction data, utilized occupancies as depression as 30% for imino acrid atoms rather and so contend with the amino acid sequence information (or lack of it in Fraser et al's example), both studies showed a deeply significant fact: They (when taken together) successfully demonstrated that even imino acrid rich sequences in the collagen molecule may adopt more than than one blazon of helical symmetry (Figures ii, 5 and half dozen). Information technology is further supported by other studies which showed variations in helical symmetry between imino acid rich and imino acid poor regions [xi]). Although neither symmetry (vii/2 or x/3) are ideally represented in either the medium resolution construction of Orgel et al 2006 [four], or the periodicities seen in the native and model Patterson office of the 00L (meridonal) series of this study for both types I and II collagen (Figure v), it would seem that at that place is evidence for the presence of closely related structures to both symmetries in molecular collagen in situ.

A possible concern that the Patterson function could be detecting common sequence repeats rather then primarily the axially repeating helical periods (Tabular array 1, Figures 5 and 6) is mitigated by information presented in Figure six. The same periodicities of interest, between 2 and viii.6 nm, are detected unchanged between the GAA/GPO models and their corresponding coordinates onto which the type I collagen sequence was substituted. That is, the periodicities of the collagen sequence do non modify the positions of periodicities seen between the different helix models. If anything, the magnitude of the signals that seem to correspond to helical symmetry periods are enhanced. Furthermore, it tin can be seen that even these models based on structures derived from high-resolution single crystal studies with the perfect helical twists, point a helical period range that is not the ideal 7/two and 10/three structures determined from fiber diffraction studies on full length collagen. Maybe their short length and more likely, the absence of the long scale molecular packing interactions seen in fibrillar collagen event this aspect of helical structure. Yet, they exercise provide u.s.a. with unproblematic, useful insight into this assay. The GAA model periodicities show a articulate increase in period over the GPO model of around 0.12 nm. A comparing between the helical model data with the experimentally obtained information for types I and II collagen bespeak that the native collagens take periodicities that are more similar to the GAA (10/3) helix model then the GPO (seven/2) model in the placement of the pitch and repeat distance parameters (Figures 5c, 6c and Tabular array 1) [33], [34]. Nosotros suspect that both helical symmetries along with other more poorly defined ones (see helix instability above and Figures 3, 5 and vi) may be found inside the fibrillar collagen triple-helical domain. If so, this may aid in explaining the diffuse, non-crystalline nature of the helical diffraction part of the collagen fiber design, as it would correspond an average of these symmetries. The fact that the high-angle layer line index of the 10/3 and 7/2 helix accounts for the same layer-lines seen in the non-crystalline diffraction [42] may not be an accident, but representative of the persistence of both symmetries in the same sample, specially when stretched or not, allowing a transformation to occur from one symmetry to another [43].

There is much yet to be resolved concerning collagen structure, even with the smashing strides in progress made recently by the diverse groups referred to in this study. We anticipate that future work volition need to increment its reliance on the use of composite data from collagen-like peptides incorporated into the context of lower resolution structural determinations from techniques such as cryo-electron microscopy and fiber diffraction to garner the better, most representative picture of collagen nature.

Acknowledgments

Cheers to the staff and scientists of the BioCAT group. Thanks also to Dr. William Dezoma for disquisitional reading and assistance in manuscript preparation. Disclaimer: The content is solely the responsibleness of the authors and does not necessarily reverberate the official views of the National Institutes of Health.

Author Contributions

Conceived and designed the experiments: JPROO AVP OA. Performed the experiments: JPROO AVP. Analyzed the information: JPROO. Contributed reagents/materials/analysis tools: JPROO AVP. Wrote the paper: JPROO AVP OA.

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