Enamel-like composite coating prepared by electrolytic ...

Enamel-like composite coating prepared by electrolytic ...

Conformational Changes of 32 kDa
Enamelin as a
2+
Function of Ca and its Interaction with
Amelogenin
Conformational
Changes of Amelogenin Following Addition of the 32 kDa Enamelin (CD Analysis)
Daming Fan, Rajamani
and Janet
Moradian-Oldak
ResultsLakshminarayanan,
and Discussion

Introduction

The CD spectrum of rP172 amelogenin has a strong negative absorbance around 201 to 203 nm, which is the characteristic of polyproline II
structure. In the absence of Ca2+, when enamelin was added to rP172 at a molar ratio of 1:100, the minimum in both pH 5.8 and pH 8.0 solutions
decreased. With increasing ratios (1:50 and 1:10), the intensity of the minima decreased further while both peaks shifted from 201 to 203 nm at pH
5.8 and from 203 to 205 nm at pH 8.0 (Fig. 5A and 5B). Meanwhile, an isodichroic point appeared at 194 nm in pH 5.8 and at 195 nm in pH 8.0
solutions, which strongly supports the conformational changes of amelogenin upon addition of enamelin. In the presence of 2.0 mM Ca 2+, the
intensity of minima also decreased with addition of enamelin. An isodichroic point at 220 nm is observed at pH 5.8 while it is at 195 nm at pH 8.0
(Fig. 5C and 5D). These conformational changes suggest that the 32 kDa enamelin interacts directly with amelogenin and the domains of enamelinCa2+ interactions are different from those involved in enamelin-rP172 interactions.

Center for Craniofacial
Molecular
Biology,
Secondary Structure
of the 32 kDa Enamelin
analyzed bySchool
CD and FTIRof Dentistry, University of Southern California, Los
Angeles,
USA
The far-UV CD spectrum of the 32 kDa enamelin in Tris buffer shows two
negative troughs at 207CA
nm with 90033,
a mean residue ellipticity
of -3.0x10
2

-1

0.5

-1

A: pH 5.8

-1.5
-2
-2.5
-3

190

A

200

210

220

230

240

250

-1.6
-2
-2.4
-2.8
190

260

0

0

-0.2

[]mrw x 10m rw x 10-3 (deg cm 2 dmol -1 )

-1

0.4

1.5
0.5
-0.5
-1.5
-2.5
-3.5
200

210

220

230

240

250

260

-0.4
-0.8

C: pH 5.8, w/Ca

-1.2

2+

-1.6
-2
-2.4
-2.8
190

200

Figure 2. Secondary structure of the 32 kDa enamelin (0.17 mg/ml in 20 mM Tris buffer, pH 7.4, 0.15 M NaCl). (A) CD spectrum.
(B) ATR-FTIR absorption difference spectrum. (C) Deconvolved spectrum. (D) Second-derivative spectrum.

3.5
2.5

-1

A
[]mrw x 10mrw x 10 ( deg cm dmol )

0.00

36

40

-3

Edman Sequence Results: LPHVPH-IPP

0.5
-0.5
-1.5
-2.5

Expression and Purification of Recombinant Amelogenin (rP172): The recombinant porcine amelogenin rP172 was expressed in
Escherichia coli, purified using RP-HPLC, and characterized as previously described 5. rP172 is analogous to the full-length porcine
amelogenin P173, but lacking the first methionine and a phosphate on Ser 16.
Spectroscopic Studies: CD measurements for the 32 kDa enamelin and its interactions with Ca and amelogenin were conducted on a
JASCO J-810 spectropoloarimeter calibrated using a 0.06% (+)-10-camphorsulfonic acid solution. Instrument optics and sample
chamber were continually flushed with 20 liters/min of dry N 2 gas. The purified and lyophilized 32 kDa enamelin was dissolved in 20
mM tris(hydroxymethyl)aminomethane (Tris) buffer at pH 7.4 with an ionic strength (IS) 0.15 (0.15 M NaCl). CD spectra were
expressed as the mean residue ellipticity, []]mrw, (deg cm2 dmol-1), and []]mrw was calculated using the equation,
[]]MRW = []] MRW/10 l c
where []] is the observed ellipticity, MRW is the mean residual weight which is defined as the M/N-1 where M is the molecular mass
(MrP172 = 19572.5; Menamelin = 32000 Da) and N is the number of amino acid residues, l is the optical path length, and c is the
concentration of the protein (mg/mL). The secondary structure contents of the 32 kDa enamelin from the CD spectra were estimated by
a FORTRAN program called CDSSTR (http://lamar.colostate.edu/~sreeram/CDPro/). The calcium binding affinity (Ka) of the 32 kDa
enamelin was determined from the fitting curve of the mean residue ellipticity at 222 nm. FTIR spectra of the 32 kDa enamelin in Tris
buffer (pH 7.4, IS 0.15) and in the presence of 2.0 mM Ca2+ were recorded by using a JASCO FT 4100 spectrometer equipped with a
fast recovery TGS detector. The interaction between the 32 kDa enamelin and rP172 in 20 mM Tris (2.0 mM CaCl 2, 0.15 M NaCl) at
various ratios was investigated by DLS at pH 5.8 and pH 8.0, respectively. DLS measurements were performed by the DynaPro99EMS/ X instrument equipped with a solid-state laser operating at 655 nm with a temperature controlled MicroSampler at 20C
(Wyatt Technologies, Santa Barbara, USA).

210

This work was funded by NIDR/NIH grants DE-13414 and DE-15644.

D: pH 8.0, w/Ca

-0.6

2+

-0.8
-1
-1.2
-1.4

210

220

230

240

250

190

260

200

210

220

230

240

250

260

Wavelength (nm)

Figure 5. CD spectra of rP172 interaction with the 32 kDa enamelin (in 20 mM Tris, 0.15 M NaCl). (A) rP172 (0.19 mg/ml),
pH 5.8; (B) rP172 (0.16 mg/ml), pH 8.0; (C) rP172 (0.18 mg/ml), pH 5.8, 2.0 mM CaCl 2; (D) rP172 (0.11 mg/ml), pH 8.0, 2.0
mM CaCl2. (rP172 (); enamelin:rP172 = 1:100 (); enamelin:rP172 = 1:50 (*); enamelin:rP172 = 1:10 ()).

220

230

240

250

260

Figure 3. CD spectra of enamelin as a function of Ca 2+. (Ca2+ concentrations (mM): 0 (); 0.05, (); 0.2 (); 0.5, (); 1.0, (+); 2.0,
(-); 10.0, (x)). (B) ATR-FTIR absorption difference spectrum in the presence of 2.0 mM Ca 2+. (C) Deconvolved spectrum. (D)
Second-derivative spectrum.

Figure 4. The calcium association constant (Ka) of
the 32 kDa enamelin calculated from the fitting
curve of the []mrw x 10mrw at 222 nm. The determined Ka of
the 32 kDa enamelin is 1.55 (0.13) x 103 M-1.

Table 1. Quantitative analysis of secondary structure of the 32 kDa enamelin
(20 mM Tris, pH 7.4, 0.15 M NaCl) as a function of Ca2+. Program: CDSSTR;
Ref. Protein Set: SP43

-1

Ca2+ Con.(mM)

-Helix

-Sheet

Turns

Unordered

a

0

81.5%

10.1%

1.5%

8.0%

0.053

0.05

68.0%

13.8%

7.4%

10.2%

0.071

The influence of the 32 kDa enamelin on the self-assembly behavior of rP172 at pH 8.0 was remarkable. Without enamelin, the self-assembled rP172
nanospheres exhibited a bimodal size distribution with one major population having a mean R H of 17.6 nm and another minor population of RH of 62.9
nm (Table 2). However, the estimated RH and MW of the nanospheres in the rP172-enamelin solution become smaller upon addition of the 32 kDa
enamelin. The average RH of nanospheres in the major population decreased to 15.1, 11.8, and 4.3 nm, respectively, which are corresponding to the
enamelin to rP172 ratios at 1:100, 1:50 and 1:10. The nanospheres detected by DLS at the elevated ratio (1:10) might be either the enamelin-amelogenin
complexes or the smaller rP172 nanospheres. We interpret these data to suggest that the particles with a mean MW of 104 kDa are likely formed by four
rP172 molecules and one enamelin, generating an enamelin-amelogenin complex, or by five rP172 molecules, giving rise to a highly disassembled
amelogenin particle. Although it is not possible to unambiguously define the composition of these particles, the DLS data clearly demonstrate partial
dissociation of amelogenin nanospheres in a dose-dependent manner with enamelin, indicating a direct interaction between these two enamel proteins.
The interaction between the 32 kDa enamelin and rP172 is likely through the tyrosyl motif at the N-terminal of amelogenin 7.
Table 2. Hydrodynamic radii (RH), molecular weight (MW), and mass distribution of particles in the
enamelin-rP172 solutions (20mM Tris buffer, 0.15 M NaCl, 2.0 mM CaCl2)
pH 5.8
enamelin : rP172

Ka = 1.55 (0.13) x103 M-1
0.2

-2

0

1

2

3

4

5

6

7

8

Calcium Concentration (mM)

9

10

a

64.5%

15.2%

6.8%

13.4%

0.062

0.5

62.6%

16.7%

5.9%

14.8%

0.046

No enamelin

1.0

58.7%

17.5%

8.1%

15.3%

0.071

1: 100

2.0

53.0%

18.0%

13.3%

16.3%

0.050

1:50

, root mean square deviation.

pH 8.0

RH

MW

Mass

RH

MW

Mass

(nm)

(kDa)

(%)

(nm)

(kDa)

(%)

2.9 0.34

41

99.4

17.6 2.2

2759

91.0

62.9 13.1

54494

9.0

2.9 0.36

41

99.6

15.1 1.7

1931

87.0

54.1 15.1

38200

13.0

11.8 0

1089

87.8

49.3 12.4

30723

11.4

196.1 17.1

778545

0.9

4.3 0

104

96.9

32.7 11.3

11770

3.0

136.0 18.2

330282

0.2

3.0 0.44

43

99.9

2+

Acknowledgements

260

Wavelength (nm)

x10-3 (deg cm2 dmol-1)

Extraction, Purification, and Characterization of the 32 kDa Enamelin: The 32 kDa enamelin was extracted following the method
described previously1 and purified by reverse-phase high performance liquid chromatography (RP-HPLC), first using a C4 column
(250x10 mm, Phenomenex) followed by a C18 column (250x10 mm, Phenomenex). The purity of extracted 32 kDa enamelin was
above 95%, as confirmed by RP-HPLC and by stains-all staining in SDS-PAGE (Fig. 1A). A blue band with a molecular weight around
32 kDa was observed and this stains-all positive response is typical to the phosphorylated glycoprotein. For protein sequence analysis
following SDS-PAGE, the gel was equilibrated in 3-[cyclohexylamino]-1-propanesulfonic acid buffer and was electrotransferred to
PVDF membrane (Millipore) at 50 volts for 30 minutes. Amino acid sequence analysis was performed at the Division of Biological
Sciences Protein Sequencing Facility at UCSD and the N-terminal sequence of the stains-all positive band was reported to be:
LPHVPH-IPP, matching the primary structure of the 32 kDa enamelin.

200

Experimental

190

[ ]mrw x 10
mrw

Figure 1. Isolation, purification and characterization of the 32 kDa enamelin. (A) Elution profile of purified
32 kDa enamelin from RP-HPLC on a C4 analytical column and stains-all staining for the 32 kDa enamelin.
(B) Edman results and amino acid sequence of the 32 kDa enamelin.

250

At pH 5.8, rP172 amelogenin has a monodisperse size distribution with a mean R H of 2.9 nm and an estimated MW of 41 kDa, suggesting dimers of
rP172 molecules. The particle distribution in the rP172-enamelin solution remained monodisperse and the size was not affected when enamelin was
added to rP172 at ratios of 1:100 and 1:50 to rP172. At a ratio 1:10 of enamelin to rP172, the size distribution of particles in the mixture is still
monodisperse but with an increase of RH to 3.8 nm. The estimated MW for the RH 3.8 nm particles is 77 kDa, which is likely due to the formation of a
complex between two rP172 molecules (based on MW 20 kDa for rP172) and one 32 kDa enamelin, indicating a direct interaction between these two
enamel proteins.

1.5

-3.5

Primary Structure of the 32 kDa Enamelin:
174
LWHVPHRIPP GYGRPPTSNE EGGNPYFGFF GYHGFGGRPP YYSEEMFEQD
FEKPKEKDPP KTETPATEPS VNTTVPETNS TQPNAPNPRG NDTSPTGTSG
QGPNPR279

240

The Effect of the 32 kDa Enamelin on Amelogenin Self-assembly (DLS Analysis)

2

32

Time (m)

210
220
230
Wavelength (nm)

-0.4

Wavelength (nm)

0.02

28

200

-1.6

-3.2

24

B: pH 8.0

-1.2

2.5

Wavelength (nm)

Abs (220 nm)

-0.8

3.5

190

(B)

-0.4

Wavel ength (nm)

2

0.04

-0.5

-3.5

-3

(A)

0

0
-1

3

2

-1

-3

2

[]mrw x 10mrw x 10 (deg cm dmol )

3

The FTIR absorption difference spectrum of the 32 kDa enamelin shows two main bands: the amide I and amide II maxima at 1655 and 1536 cm 1
respectively, and two minor bands at 1741 and 1582 cm -1 (Fig. 2B). The amide bands contain a number of absorptions as revealed by the
deconvolution and second-derivative spectra presented in Fig. 2C and 2D, respectively. It further showed a shoulder at 1671 cm -1 and a weak
absorption at 1614 cm-1 in the amide I region and a peak at 1513 cm-1 in the amide II band from the deconvolution and second derivative analysis.
The major amide I component (1655 cm-1) can be assigned to -helical and/or random coil structures. Other amide I components at 1614 cm -1
region are associated with -sheet while the shoulder at 1671 cm -1 is assigned to an anti-parallel -sheet. The component at 1741 cm -1 is attributed
to stretching vibration from the COO- groups while the band at 1582 cm-1 could be related to asymmetric stretching vibration of COO- groups.

[]mrw x 10m rw x 10 ( deg cm dmol )

We applied circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy to study secondary structural preferences of the
32 kDa enamelin in the absence and presence of calcium ions. We further used CD and dynamic light scattering (DLS) measurements to
investigate amelogenin-enamelin interactions, in various ratios of these two proteins at pH 5.8 and 8.0 and in the presence of Ca 2+. A
recombinant porcine amelogenin (rP172) was used. The knowledge of calcium effect on the conformational changes of the 32 kDa
enamelin and the cooperation between enamelin and amelogenin will contribute to the understanding of enamelin structure and function
in enamel biomineralization.

deg cm dmol and at 220 nm with -2.3x10 deg cm dmol , and a positive maximum around 192 nm of 3.3x10 deg cm dmol (Fig. 2A), which
are characteristics of the secondary structure of proteins having a high content of -helix. Quantitative estimation for the secondary structural
fractions of the 32 kDa enamelin by the program CDSSTR showed that it has 81.5% -helix content, 10.1% -sheet, 1.5% -turns and 8.0%
unordered structure (Table 1).
-1

[]mrw x 10m rw x 10-3 (deg cm2 dmol-1 )

Objectives and Strategy

3

2

[]mrw x 10mrw x10 -3 (deg cm 2 dmol -1 )

Enamelin, a phosphorylated glycoprotein that constituents a small percentage of the extracellular matrix (<5%), plays an important role in enamel formation. In developing porcine enamel, enamelins have been isolated with molecular weights of 25, 32, 45, 89, 142 and 155 kDa Among them, the 32 kDa enamelin is the most stable fragment of 186 kDa enamelin (extending from Leu 174 to Arg279) and has been studied for its structural and functional properties 1. The 32 kDa enamelin is hydrophilic and acidic with a pI 3.2, and is rich in proline (18.8%), glycine (12.3%), threonine (10.4%), and glutamic acid (9.4%) 2. It has two phosphorylated serines (Ser191 and Ser216) and three glycosylated asparagines (Asn245, Asn252 and Asn264)2. Furthermore, the 32 kDa enamelin was shown to have high affinity to bind to apatite crystals3, highlighting relevant functional properties of enamelins in controlling crystal nucleation or growth. The addition of 32 kDa enamelin onto amelogenins also promoted the nucleation of apatite crystals 4. Despite the advances in understanding enamelin primary structure, the secondary structural properties of enamelin and its interactions with ligands are still largely unexplored. 1:10 Conformational Changes of the 32 kDa Enamelin as a Function of Ca 2+ The CD spectra show that sequential addition of CaCl2 (0.05-10.0 mM) resulted in a progressive decrease in intensity of the minima at 208 nm and 220 nm and the maximum at 192 nm (Fig. 3A). When Ca2+ increased from 0.05 to 2.0 mM, the troughs are slightly blue-shifted to 208 nm with decreased intensity -1.4x103 deg cm2 dmol-1 and to 221 nm of -1.0x103 deg cm2 dmol-1. This blue shift is also observed for the positive band from 192 nm to 193 nm at 2.0 mM Ca2+. At higher concentrations (5.0 and 10 mM Ca2+), there is no further conformational change. Therefore, the 2.0 mM Ca 2+ is a saturation point of calcium effect to the 32 kDa enamelin conformation. A careful examination of the CD spectra reveals the presence of an isodichroic point at 200 nm, indicating a two state equilibrium between a-helix and -sheet conformations and a strong support to the conformational changes of the 32 kDa enamelin as a function of Ca2+. CDSSTR analyses indicate that at an initial 0.05 mM Ca 2+, the -helix content of enamelin decreased from 81.5% to 68.0% with a concomitant increase of -sheet from 10.1% to 13.8% and -turns from 1.5% to 7.4%. With higher Ca 2+ concentrations, the -helix content of enamelin decreased progressively while the -sheet, the -turns, and other structures increased simultaneously (Table 1). Such quantitative analysis clearly demonstrates that Ca2+ binding to the 32 kDa enamelin decreased its -helix content but increased its sheet, -turns, and other structures, suggesting a preference of -sheet conformation of the enamelin in the presence of Ca 2+. The calcium association constant (Ka) of the 32 kDa enamelin was calculated from the fitting curve of the []] mrw at 222 nm (Fig. 4). The determined Ka of the 32 kDa enamelin is 1.55 (0.13) x 103 M-1, which is in good agreement with the value of 5.2x103 M-1,6 indicating a weak affinity of enamelin to calcium ions. In the presence of 2.0 mM Ca2+, FTIR difference spectrum of the 32 kDa enamelin solution apparently shows the broadening of amide I band, the weakening of the amide II region and the shift of absorption of the COO - groups (Fig. 3B). The deconvolution and second-derivative spectra of enamelin with Ca2+ (Fig. 3C and 3D) show that in addition to the shoulders at 1672 cm -1 and 1614 cm-1, a new absorption appears at 1638 cm-1, which is associated with -sheet. The presence of the new absorption and the broadening of the amide I band are in good agreement with the increase of sheet conformation upon the addition of Ca2+ in the CD study. The intensity of the amide II band weakened and it became broad at 1539-1543 cm -1 at 2.0 mM Ca2+. It is also noticeable that the intensity of the asymmetrical vibration of COO - group at 1582 cm-1 is significantly reduced while the symmetrical stretching mode shifts to 1749-1765 cm-1 and becomes broadened, clearly as a result of Ca2+ binding to COO- groups. 3.8 0.51 77 99.4 Conclusions CD and FTIR studies showed that the 32 kDa enamelin has a high content of -helix and undergoes conformational transition to -sheet as a result of Ca2+ addition. The 32 kDa enamlelin has a weak binding to Ca2+ through the carboxylate groups. DLS analyses demonstrated that the 32 kDa enamelin has a profound effect on amelogenin self-assembly at pH 8.0, giving rise to partial dissociation of rP172 nanospheres in a dose dependent manner. We interpret our data to suggest that the 32 kDa enamelin interacts with amelogenin, most likely through the Tyr-rich motif at the N-terminal. We further suggest that the 32 kDa enamelin and amelogenin may cooperate to control crystal growth during the post-secretory stage of amelogenesis. References 1. 2. 3. 4. 5. 6. 7. Yamakoshi, Y. Calcif. Tissue Int. 1995; 56, 323-330., Brookes et al 2002, Connect Tissue Res, 43: 477-481. Hu, C. C., and Y. Yamakoshi. Crit. Rev. Oral. Biol. Med. 2003; 14, 387-398. Tanabe, T., T. Aoba, E. C. Moreno, M. Fukae, and M. Shimizu. Calcif. Tissue Int. 1990; 46, 205-215. Bouropoulos, N., and J. Moradian-Oldak. J. Dent. Res. 2004; 83, 278-282. Ryu, O. H., A. G. Fincham, C. C. Hu, C. Zhang, Q. Qian, J. D. Bartlett, and J. P. Simmer. J. Dent. Res. 1999; 78, 743-750. Yamakoshi, Y., T. Tanabe, S. Oida, C. C. Hu, J. P. Simmer, and M. Fukae. Arch. Oral Biol. 2001; 46, 1005-1014. Ravindranath, R. H., J. Moradian-Oldak, and A. G. Fincham. J. Biol. Chem. 1999; 274, 2464-2471.

Recently Viewed Presentations

  • Codes and Conventions of Fashion Magazine

    Codes and Conventions of Fashion Magazine

    Street wear is a distinctive style of street fashion, rooted in West Coast. It has elements of hip-hop, urban-culture, casual and modern haute couture fashion. Street fashion is generally associated with youth culture, and is most often seen in major...
  • NATS 101 Section 6: Lecture 2

    NATS 101 Section 6: Lecture 2

    Climate data and models What they show us and how they can be used in planning BY Henri TONNANG From the IPCC AR5 report: Climate System: "The climate system is the highly complex system consisting of five major components: the...
  • Gesu&#x27; ci ensegnato a pregare

    Gesu' ci ensegnato a pregare

    With a humble heart, and relaxed mind, focus on the Creator; allow GOD to be The only subject in your mind while reading the following… Dear LORD I thank you for this day; I thank you for being able to...
  • Lecture 03  General aspects of molecular signaling pathways

    Lecture 03 General aspects of molecular signaling pathways

    The extracellular N-terminal region exhibits a variety of conserved elements including immunoglobulin (Ig)-like or epidermal growth factor (EGF)-like domains, fibronectin type III repeats, or cysteine-rich regions that are characteristic for each subfamily of RTKs; these domains contain primarily a ligand-binding...
  • Green Algae and Land Plants - Socorro Independent School District

    Green Algae and Land Plants - Socorro Independent School District

    A single common ancestor gave rise to all of the green algae and land plants. Green algae are paraphyletic. The green algae include some but not all of the descendants of a single common ancestor. Zygnematophyceae is the closest living...
  • Doing Digital History - Rice University

    Doing Digital History - Rice University

    More services? (e.g. digitization, consulting on digital tools, archiving papers & data) What else? UCL recently launched a digital anthro. MA This slide deck, which was prepared for a presentation to anthropology faculty & grad students at Rice, includes slides...
  • Integral Calculus - Ms Gugger&#x27;s Classes 2016

    Integral Calculus - Ms Gugger's Classes 2016

    Surds. Equating. Powers. Rotation. Anticlockwise. Key Notation. Integration by substitution. Finding integrals in the form of ??+???? where ?∈? ... Find an antiderivative of 4?+1?2+1 by first expressing it as partial fractions. ...
  • Hybu Ymarfer Ieithyddol Addas Promoting Language Appropriate ...

    Hybu Ymarfer Ieithyddol Addas Promoting Language Appropriate ...

    Weithiau mae cost i ddefnyddio y mesurau a thro arall does dim cost, ond mae'r perchnogion hawlfraint eisiau gwybod bod ymchwilwyr yn defnyddio y mesurau. Os ydy'r mesur ar gael i'w ddefnyddio am ddim, mae linc i'r mesur o wefan...