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Native Collagen and Its Structural Fulcrum Through Site Specific Hydration Topology Map Akhila Viswan, and Neeraj Sinha J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b05185 • Publication Date (Web): 07 Aug 2017 Downloaded from http://pubs.acs.org on August 8, 2017

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The Journal of Physical Chemistry

Native Collagen and its Structural Fulcrum through Site Specific Hydration Topology Map Akhila Viswan1,2 and Neeraj Sinha*1. 1

Centre of Biomedical Research, SGPGIMS Campus, Raebarelly Road, Lucknow – 226014 INDIA 2

Faculty of Engineering and Technology, Dr. A. P. J Abdul Kalam Technical University, Lucknow 226021, INDIA

*Corresponding Author E-mail: [email protected] , [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT: Structural account of collagen in its native environment is limited and water mediated hydrogen-bonding network as its stability analogue needs to be contemplated site specifically. We present in this article that through natural abundance

13

C chemical shift

anisotropy (CSA) measurement of collagen in native state (inside bone matrix), structural and mechanistic insight of water-mediated hydrogen bonding network can be achieved. The

13

C

CSA of backbone and side chain residues of collagen, perturbed under dehydration and H/D exchange conditions can be measured. The changes in 13C CSA values due to perturbation of water content have resulted in determining the site-specific accessibility of water molecules. Further hydration topology map (HTM) representing water accessibility of native collagen has been generated based on changes in

13

C CSA values. Our results signifies the water

accessibility of proline/hydroxyproline carbonyl region is larger compared to hydroxyproline Cγ site and open up of new avenues for collagen structure determination in its native environment.


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1. INTRODUCTION Collagen has always been a bottom line of research but the structural clues to the coiled coil triple helix has been mostly confined around extracted collagen1, model peptides2 and its molecular dynamic simulation3. Vast data pouring in from x-ray diffraction4 and neutron diffraction data gives a static picture and overlooks the dynamical influence of amplitude and orientation dependent interactions in native environment. Native collagen is different from in vitro grown tissues and other mimics of collagen for drawing subtle conformational changes reflected in its varied functions in extracellular matrices (ECM). In vitro tissue models5 depict structural refinement but at the cost of fidelity of native state. Therefore, native collagen always holds a befitting choice for structural interpretation in terms of monitoring ECM diseases, tissue engineering and designing of related tissue allografts. Collagen is universal yet unique structural protein that gathers significant structural rationale due to its stability and functional flexibility.6 The prevalence of Gly-X-Y tripeptide repeat (X and Y is mostly occupied by proline and hydroxyproline) makes this coiled coil triple helix distinct from other secondary motifs.7 Among the different extracellular matrices that houses collagen, bone is one such hardest composite biomaterial that attains its resilience due to the organic phase comprising mainly type 1 collagen.8 The unique mechanical properties of bone and other connective tissues depend on the conformation and assembly of collagen9 and its interaction with water.10 In this direction, water mediated changes with emphasis on dehydration and its impact on the strength and toughness of bone has been the basis of many studies, which clearly indicates the role of water in determining the mechanical behavior of bone.11-12 Many alternative models and theories have surfaced governing the



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water mediated bridges13 and its interaction with hydroxyl group of hydroxyproline14-15 in conferring structural integrity of collagen but still there is scarcity of literature in its native state.16-17 Water does provide the microenvironment stabilizing collagen in diverse ECM18 so its variation or perturbation will definitely reflect in collagen assembly. Water dependent changes has been reported but site specific changes needs to be mapped by more sensitive parameter reflecting functional relevance of residue mediated interaction in imparting stabilization. Literature data is deficit and no substantial structural evidence of native collagen is reported in terms of site-resolved changes sensitive to water bridges. For this purpose, chemical shift anisotropy (CSA) through solid-state nuclear magnetic resonance (ssNMR) is sensitive and insightful probe, drafting the underlying site resolved iso-aniso correlation in collagen at natural abundance in addition to monitoring local water mediated perturbation. ssNMR provide advantage over other spectroscopic techniques in terms of elucidating and exploring such structural studies of fibrous and amorphous system like collagen19-21 to its dynamic molecular connectivity’s with water22-23. By selectively introducing

13

C CSA recoupling24 which relates to both secondary structure25 and hydrogen

bond interactions26 chemical shielding along three principal axes (σ11, σ22 and σ33) of chemical shift tensors (CST)27 can be measured.CST restraints have also been proven to validate atomic structures and protein structural refinement directing further de novo structure determination.28-29 Our present study highlights that the collagen assembly is stabilized by hydrogen bonds, which interconnect triple helices through water-mediated bridges giving the secondary structure its unique flexibility and conformation. Therefore, any changes or disruption of hydrogen bond will affect the triple helical backbone and dynamics reflecting in its local environment. Therefore, in our study we have tried to map the collagen structural changes by targeting the water mediated bridges and the corresponding local environment changes which 4 ACS Paragon Plus Environment

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is correlated to CST by generating a hydration topology map (HTM). CST in complex system like native collagen at natural abundance in terms of hydration topology and stability has not been reported so far. Therefore, in our study we determined residue specific

13

C CST of

multiple site-resolved changes of native collagen under the state of surrounding water bridges showing an imperative constraint for further structure validation and accuracy. 2. EXPERIMENTAL SECTION 2.1 Sample preparation: All the ssNMR experiments were setup on cortical femora bone of Indian Goat (Capra hircus, 2-3yrs old) obtained from local slaughterhouse. The intact bone was filed with scalpel into small size flakes(Figure S1), which morphologically resembled intact bone as it was not subjected to any pre-treatment or processing since grinding alters the uniformity and water content of the intact sample.16 Also the

13

C spectra of bone flakes (Figure S3) were found

identical to intact bone spectra30 reported earlier, representing all the collagen residues. The sample was packed inside 3.2 mm Zirconium rotor for further ssNMR experiments. Six bone samples were prepared for the study including fresh hydrated one, which was not subjected to any lyophilization thereby retaining its hydration level, and the other three samples were freeze dried in lyophilizer for 5min, 10min and 30min. The other two samples were dipped in D2O for 48 hrs and 72 hrs respectively in shaker incubator to allow its maximum exchange with D2O and consecutive freeze-drying in lyophilizer with immediate recording the ssNMR spectra. 2.2ssNMR Experimental parameters: All ssNMR experiments were carried on 600 MHz NMR spectrometer (Avance III, Bruker Biospin, Switzerland) operating at 600.154 MHz for 1H and 150.154 MHz for 13C frequencies



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equipped with Bruker 3.2mm Efree probe. All one dimensional (1D) and two dimensional (2D) spectrum were set at 6.5 kHz Magic angle spinning (MAS)

using Bruker MAS

pneumatic unit with an accuracy of ±2Hz. Pulse length was calibrated to 3.12 µs for 1H π/2 pulse with 5s recycle delay. For

13

C 1D CPMAS, ramp cross polarization sequence with

spinal-64311H decoupling of 86 kHz and 1.0ms contact time. CPTOSS pulse was tuned to 8.5 µs13C π pulse and 6.5kHz MAS. 2D SUPER32 pulse sequence (Figure S4) was employed for CSA recoupling. Signal averaging time for each 2D SUPER spectrum was 18hrs with accumulation of 512 transients and 1k data points. The evolution period corresponded to 32 t1 point in the indirect dimension with rotor synchronized rf pulses and dwell time tuned to 8.4µs in accordance with 0.155 scaling factor. SUPER is iso-aniso correlation experiment wherein MAS averaged

13

C CSA recouples during the evolution period and

13

C isotropic

chemical shift during the detection period (Figure S5). 13C CSA was recoupled under MAS using a train of rotor synchronized 360° rf pulses. The

13

C CSA was extracted from the

recoupled line shape using the well-resolved isotropic shift of the collagen in the direct dimension. 2.3 CSA Simulation and Data fitting: The experimentally determined CSA powder pattern corresponding to each isotropic shift was extracted and imported in matNMR33 supported by MATLAB (The MathWorks Inc.) for generating the best fit spectra. Numerical simulations were based on SIMPLEX34 algorithm. The simulation parameters were set identical to the experimental values. The IUPAC convention is followed in reporting the three principal values of CSA. The following tensor components σ11, σ22 and σ33 for each peak were calculated with identical experimental condition. The values of tensor components showed a good agreement between experimental and simulated spectra. The three principal values of CSA tensors were calculated for all the six samples subjected to different dehydration conditions and H/D exchange. 6 ACS Paragon Plus Environment

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3. RESULTS AND DISCUSSION The most striking feature of collagen triple helix is its tripeptide repeat (glycine,proline and hydroxyproline) unit and water mediated H-bonds network. Any change in water content and H – bonding network will affect the collagen structural integrity. When the water content is perturbed, it influences the collagen structure with tripeptide being more susceptible to such changes due to its abundance and distinctive geometry.35 Therefore, we took bone sample and subjected it to dehydration and H/D exchange and the respective change in the H-bond were monitored by relative

13

C CSA measurements. Thus, changes in

13

C CSA values due to

dehydration and H/D exchange will be an indicator of water accessibility at corresponding sites of collagen. 3.1 Water distribution in bone In the present study, six compact bone samples were prepared by filing into flakes resembling intact bone (Figure S1) and subjecting to different sample conditions to study the effect of water content and the associated changes due to dehydration and H/D exchange. The water content of the sample was varied by freeze-drying for different time interval with simultaneous measurement of dehydration by recording one dimension (1D) proton (1H) spectra. The native state of the sample was also monitored by comparing with the 1H spectra of intact bone. The 1D 1H spectrum of native collagen inside bone is shown in Figure 1a, showing the relative distribution of bound water and mobile water of the hydrated intact sample. The peak observed around 1.2 ppm in 1H spectra is assigned to triglyceride as reported by Mroue et al36 stating its origin to be bone marrow or blood vessels which is found abundant in trabecular bones as NMR signals from phospholipid and cholesterol are usually not detected in cortical bones. The water is entangled in the bone matrices as pore water (mobile water) occupying the Harvesian-lacunocalicular system and bound water



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associated with both collagen and hydroxyapatite crystal in the inorganic phase. The relative distribution of water and its entrapment in the bone matrices is changed when the sample is dried for 5min, 10min and 30min and when exchanged with D2O for 48hrs and 72hrs(Figure S2).Dehydration resulted in rearrangement of hydration network shown in the ratio of mobile water to bound water with pronounced effect seen in mobile water content when the sample is dried for 30min.In case of H/D exchange samples, the exchange effect is seen for both mobile and bound water contrary to dehydration samples where mobile water is mostly affected. The sharp peak in the 1H spectrum observed in H/D exchange samples is due to residual un-exchanged mobile water. The H/D exchange phenomena in bone is employed not only to quantify pore water and collagen bound water, but its interrelated microdyanamical properties can also be studied.37-39 Long time deuteration has been found to affect the bound water moieties which is elaborated using Raman spectroscopy on cortical bone.40

A corresponding

13

C 1D spectrum of collagen is shown where aliphatic region shows the

prominence of hydroxyproline Cγ (HypCγ) at 71.1ppm, proline Cα (ProCα) at 60.1ppm and glycine Cα (GlyCα) at 43.2ppm residues also with occurrence of alanine Cα (49.7ppm), Hyp Cδ(54ppm), Pro Cδ(48.1ppm), citrate (76.5ppm) and other polar residues like glutamate (Figure 1b). The residues of values.16,

30

The

three

13

C collagen spectra are assigned using the previous literature

characteristic

peak

mostly

dominated

by

glycine

and

proline/hydroxyproline carbonyl region resonating at 169ppm and 171.7ppm/174.6ppm respectively are distinctive of hydrated bone sample (Figure 1c).16,

41-42

Tropocollagen

constitutes approximately 33% Glycine, 20-25% Proline/Hydroxyproline and 10-12% alanine20 and as reported by Saito et al it is reasonable to assign the resolved 13C signals of collagen fibrils to the abundance of these residues.43 3.2 Dehydration introduced changes in 13C collagen spectra


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Dehydration induced changes were monitored in the

13

C 1D spectra of hydrated collagen

sample with notable difference observed in the line broadening and the signal intensity when sample condition was varied(Figure S3).Line broadening was detected in HypCγ, Ala Cα, HypCβ and Ala Cβ resonances and the sharp signal intensity was observed for the H/D exchanged sample. The merging of the peaks in the carbonyl region compared to the three distinctive peak of hydrated sample is indicative of the loss of water whereas H/D exchange resulted in the clear resolved peaks of Pro/Hyp C’ and Gly C’.

13

C chemical shift are

conformation dependent with backbone carbon being more sensitive intrinsic probe for conformation characterization. However, due to the contribution of various factors, the apparent change in the 1D

13

C line width and line broadening cannot be interpreted for

possible conformation dependent changes. Collagen is stable yet flexible so any local change will reflect in their diverse functional aspect, which is to be further monitored by sensitive probe like 13C CSA tensor measurement. 3.3 CSA powder pattern of native collagen Therefore, 13C line shape analysis and its correlation to water content was further probed by measuring the principal values of CSA which is more sensitive to local electronic environment44 for mapping the subtle variation in the interrelated hydrogen bonding interactions.13C 1D isotropic values helped us to measure the CSA of multiple sites which was well resolved by SUPER32 (Figure S4), a robust and efficient method for the site specific determination of principal components of CSA. We used CSA recoupling to obtain sitespecific insight into hydrogen bonding and its mediated perturbation in the collagen triple helix. CSA recoupling resulted in reintroducing the undistorted CSA powder pattern in the indirect dimension for both aliphatic and the carbonyl region and the resolved

13

C isotropic

chemical shift of collagen sites in the direct dimension (Figure S5). The accurate determination of tensor parameters from the powder pattern depended on the best-fit values 9 ACS Paragon Plus Environment


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generated by numerical simulation. The principal components σ11, σ22 and σ33 were determined based on the IUPAC convention where σiso corresponds to (σ11+σ22+σ33)/3. The CSA tensor values of the backbone carbonyl region, aliphatic region and side chain residues have been determined and shown in Table 1. The matNMR SIMPLEX algorithm is used to obtain the best-fit values following Gaussian line broadening and error values within the experimental limit (Figure2a-j). Table 1: Chemical shift tensor components σ11, σ22 and σ33 of backbone carbonyl, aliphatic residues under different conditions and side chain residues of hydrated native collagen 13

C CSA*(ppm) of collagen backbone and side chain

HydroxyprolineCγ

σ11

σ22

σ33

45

78

90

44

79

90

36

77

100

46

79

90

43

73

100

51 σ11

75 σ22

88 σ33

35

65

81

32

64

83

31

65

84

28

61

90

32

64

83

27 σ11

70 σ22

82 σ33

21

31

74

15

42

73

Hydrated Dry 5 min Dry 10 min Dry 30 min H/D Exchange 48 hrs H/D Exchange 72 hrs Proline/HypCα Hydrated Dry 5 min Dry 10 min Dry 30 min H/D Exchange 48 hrs H/D Exchange 72 hrs Glycine Cα Hydrated Dry 5 min


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Dry 10 min 23

46

61

17

47

63

21

40

69

11 σ11

37 σ22

81 σ33

117

163

226

116

159

228

121

167

225

110

159

237

114

171

221

125 σ11

150 σ22

229 σ33

107

155

252

117

166

233

113

174

227

109

175

234

126

172

215

122 σ11

181 σ22

211 σ33

121

172

231

122

171

229

113

161

245

116

168

233

102

174

248

103 σ11

173 σ22

247 σ33

Dry 30 min H/D Exchange 48 hrs H/D Exchange 72 hrs Glycine C’ Hydrated Dry 5 min Dry 10 min Dry 30 min H/D Exchange 48 hrs H/D Exchange 72 hrs Proline/Hyp C’ Hydrated Dry 5 min Dry 10 min Dry 30 min H/D Exchange 48 hrs H/D Exchange 72 hrs Proline/Hyp C’ (174ppm) Hydrated Dry 5 min Dry 10 min Dry 30 min H/D Exchange 48 hrs H/D Exchange 72 hrs Side chains



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Citrate

66

72

90

Alanine

24

51

73

ProlineCδ

21

50

72

HydroxyprolineCδ

29

59

73

*

Errors in the measurements of CSA are ±3 ppm for unprotonated carbonyl group, ±1 ppm for

aliphatic groups 3.4 Chemical shift tensors of collagen residues The

CSA

tensor

values

of

the

carbonyl

region

corresponding

to

glycine,

proline/hydroxyproline region were measured where dash line represents the best fit spectra and bold line represents the experimental one (Figure2h-j). The variation in the individual tensor components under different set of conditions is indicative of structural changes due to the hydration perturbation (Figure S9-S11). Such changes in tensor values are also descriptive of the spatial dependence on hydrogen bond distance and dihedral angle. Although alanine and glutamate carbonyl peaks will resonate around same region but since their abundance is much less compared to others thereby, their CSA measurement by this method is rather difficult to resolve. The probable behaviour of carbonyl region and its electronic environment when subjected to dehydration and H/D exchange is monitored by CST change. The carbonyl CST will tend to be same for other amino acids also but the most likely changes are being reflected by the abundant amino acids. Similarly, values were determined for Cα backbone aliphatic regions (Figure 2b, 2g) that are strongly correlated to backbone torsion angle. Values of 13Cα CSA and its changes corresponding to rearrangement of hydration network on dehydration and H/D exchange (Figure S7, S8) are important determinants of backbone geometry. Backbone carbonyl and Cα CSA as a function of three


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principal elements are fundamental CST restraints in structural studies and helpful in predicting secondary structure and interlinked hydration dynamics.45-47 HypCγ, is post translational modified with hydroxyl group at its gamma position and has always been most intriguing in the study of collagen due to its role in stabilization.41 The tensor component of HypCγ (Figure2a) was determined to observe for the changes mainly due to the Hyp hydroxyl group in stabilization of triple helix through its water-mediated interactions (Figure S6). Its tensor parameters thus holds substantial in determining the structural role of water in collagen. The tensor values of collagen side chain Pro Cδ (Figure2c), Ala Cα (Figure2d), HypCδ (Figure2f), and citrate (Figure2e) were measured with their respective principal components shown in the Table1. Tensor values of side chain residues complement the backbone conformational data by reflecting the underlying chemical trend and indicating the nature and directionality of bonding. 3.5 Hydration topology map CST of different residues were measured and the best fit values of each residue with respect to the CST difference of the corresponding residues under different conditions were used to obtain the , and values using the Pearson’s coefficient equation as shown below.

= ∑

−

−

= ∑

(1)

(2)

− (3) = ∑

Where, σ11, σ22 and σ33represents the tensor components at different conditions, n represents the number of conditions employed



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i represents different conditions of dehydration (5min,10min and 30min) and H/D exchange (42hrs,72hrs)

The above equation measures the relative change of each CSA component for every residue in collagen under different conditions thus providing a direct measure of the effect of hydration and H/D exchange at each site with respect to water mediated structural changes. Hence, such measure can be termed as Hydration Topology Map (HTM) of collagen. The best-fit tensor components of different sites studied across independent conditions of dehydration and H/D exchange resulted in obtaining the HTM (Figure3a), reflecting the correlation of CST perturbation with hydrogen bond network. The Figure 3a shows the plots of , and at different sites of collagen. It shows that if dehydration and H/D exchange affects the hydration network of collagen it will result in change in the CSA values as shown in color bar of the HTM. Red color indicating more impact at component of Pro/Hyp C’, while yellow color reflecting change in the component with green color somewhat less affected and blue color indicative of stability or least affected sites to H-bond changes. Thus, HTM demonstrates that the sites, which are more receptive to changes in water mediated hydrogen bonding, will undergo CST perturbation at either component. The above results reveals Pro/Hyp C’ tensor component to be more prone to H-bond changes while HypCγ more stable to different conditions of dehydration and H/D exchange. Thus, changes in CSA tensor components due to perturbation of water will be an indicator of water accessibility at different sites. Hence, quantitative measure of such CSA component change will be an indicator of native collagen hydration topology. HTM was generated to determine the different water bridges present in the collagen and at the interface of collagen and hydroxyapatite (HAP) (Figure3b) which are more prone to affect by dehydration and H/D exchange. Figure 3b displays the loosely bound water that


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is mostly present at the surface of collagen and occupies the space between collagen and mineral phase. The tightly bound water found inside collagen triple helix is shown in Figure 3b as direct H-bonds (between CO-NH) and inter and intra chain H-bonding (involving CO and Hyp-OH mediated).35, 48-49 The contribution from other water bridges involving amide linkages, which are generally present in imino poor regions with different degree of stabilities are equally important.35, 50 But the above method poses limitation in measuring such water bridges with propensity of amino acids at Xxx other than Proline/Hydroxyproline is low at natural abundance. HTM represented the water accessible sites of all possible residues of collagen in native state at natural abundance. The HTM using CST restraints provided lines of evidence supporting the principal role of HypCγ in imparting stabilization, which thereby forms the mainstay of fibrillar collagen. Mild dehydration resulted in affecting the loosely bound water present at the interface of collagen and HAP and thereby the backbone carbonyl region of Pro/Hyp was more susceptible to dehydration induced changes.30 The hydroxyl group of HypCγ mediated water bridges were strong enough to be least affected by dehydration and H/D exchange thus validating their role in stabilization of collagen helix under mechanical load.35, 48The tensor component more prone to vary can depict the electronic environment and its distortion under conditions of stress and damage due to the proximity of certain hydration sites and its interaction with collagen residues. Through the HTM of collagen in native state, we have been able to map the subtle changes in principal components of multiple sites each holding information guiding to the conformational stability of collagen under higher order hydration network. 4. CONCLUSION



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Measuring the chemical shift parameters in collagen due to dehydration is a sensitive measure to probe the sites more prone to affect by rearrangement of hydration network.We have presented here the underlying catalogue of tensorial information corresponding to the changes in the distribution of water and its interaction with native collagen. Hydrogen bonding and its effect on

13

C CSA, which is more sensitive to hydrogen-bonded, will be

significant in highlighting the structural related changes under dehydration as well as tropocollagen assembly stabilized by water bridges. This is the first time that the CSA trend and its correlation with hydration milieu in native state collagen in natural abundance have been shown. Tonsorial information of such complex bio system in terms of CSA in native state is underrepresented compared to isotropic chemical shift but is descriptive and interpretive when probing unusual geometric structures. Such data is lacking in literature and thus holds paramount importance in studies of structure refinement when combined with other theoretical and related x-ray diffraction generated data. Structural accuracy will be helpful in interpreting bio mineralization of collagen in various ECM having viscoelastic and biomechanical properties resembling the native state. Native collagen and the related changes in CST restraints provide a true picture of systems dynamic changes corresponding to water distribution. Subsequently the study is pivotal to understand the residue specific and extrinsic factors (age and diseases) related affects for further structure quality and refinement.


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ASSOCIATED CONTENT: Supporting Information (SI): The Supporting Information contains from FigureS1-S11. FigureS1 shows picture of powder bone flakes from intact bone, FigureS2 shows distribution of mobile water and bound water inside bone in hydrated native state and under different conditions of dehydration, FigureS3 shows

13

C 1D CPTOSS spectrum of collagen showing

the aliphatic and carbonyl region of collagen in hydrated native state and under different conditions of dehydration, FigureS4 shows pulse sequence of SUPER¸FigureS5 shows recoupled CSA lineshape for aliphatic and carbonyl region of collagen in the indirect dimension, FigureS6- FigureS11 shows best fit of experimental CSA powder pattern of HydroxyprolineCγ,

Proline/Hydroxyproline

Cα,

Glycine

Cα,

Glycine

C’,

Proline/Hydroxyproline C’ at 171.7 and 174 ppm respectively recoupled from 2D SUPER with chemical shift tensor values denoted by σ11, σ22 and σ33.

AUTHOR INFORMATION: Corresponding Author E-mail: [email protected] , [email protected] Notes: The authors declare no competing financial interest. AUTHOR CONTRIBUTIONS: AV performed ssNMR experiments and data analysis. NS performed the data analysis, interpretation of data and conceptualization of the project. Both authors gave approval to the final version of the manuscript. ACKNOWLEDGMENTS The authors gratefully acknowledge grant from SERB, Department of Science & Technology, INDIA(EMR/2015/001758) for financial support.



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Figure Captions: Table 1: Chemical shift tensor components σ11, σ22 and σ33 of backbone carbonyl and aliphatic residues and side chain residues of native collagen under different conditions Figure1: a)1H 1D spectra showing the distribution of mobile water and bound water in bone b)13C 1D spectra showing the aliphatic region of collagen, c)

13

C 1D spectra showing the

carbonyl region of collagen Figure 2: a-g) CSA tensor components of backbone aliphatic region and side chain residues, h-j)CSA tensor components of carbonyl region Figure 3:a) Hydration topology map of different residues of native collagen b) Water bridges in the collagen triple helix, HAP and at the interface of collagen and HAP,


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Figure1: a) 1H 1D spectra showing the distribution of mobile water and bound water in bone b)13C 1D spectra showing the aliphatic region of collagen, c) 13C 1D spectra showing the carbonyl region of collagen 208x80mm (300 x 300 DPI)

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Figure 2: a-g) CSA tensor components of backbone aliphatic region and side chain residues, h-j) CSA tensor components of carbonyl region 192x317mm (300 x 300 DPI)



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Figure 3: a) Hydration topology map of different residues of native collagen b) Water bridges in the collagen triple helix, HAP and at the interface of collagen and HAP 622x310mm (300 x 300 DPI)


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TOC Graphics 71x43mm (300 x 300 DPI)


Native Collagen and Its Structural Fulcrum through ... - ACS Publications

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