패각의 결정내부단백질 2차구조의 정량적 해석

김용완,윤은선,최청송

서강대학교 화학공학과

A Quantitative Secondary Structure Analysis of the Intracrystalline Protein extracted from Oyster Shell.

Yong-Wan Kim, Eoun-Sun Yun, Cheong-Song Choi

Dept. of Chemical Engineering, Sogang University

1. Introduction

Aragonite and calcite are the two carbonate skeletons most frequently encountered in biological systems. These biogenic minerals have the unique mineralogy quite different from those found in nonbiological precipitates[1]. It is known that CaCO3 polymorphs constitute 98 to 99% of the inorganic material of oyster shells. Also it was reported that aragonite was formed easily from a solution at a temperature above 50℃. It is likely to more easily induce the nucleation of the thermodynamically more stable polymorph calcite[2]. The aragonite, however, has been readily found in the deposited minerals.

A number of experiments have been made to inspect the mechanisms of inorganic crystallization at mineralized tissue. The biological approaches have been to isolate the protein components from organisms and examine the formation of calcium salts in the presence of these components[3]. The difficulty with these studies is that the exact nature of the interaction between the minerals and the shell proteins is not well understood. One major reason is the difficulty of analyzing biomineralization processes in their native state[4]. Biomimetic systems have indicated the importance of electrostatic binding or association, geometric matching(epitaxis), and stereochemical correspondence at inorganic-organic interfaces. Although these methods appear to be plausible, they have not provided adequate explanations for the oriented crystallization in biological systems. Yet we have little understanding of crystal science in terms of bonding and reactivity of organized structures.

In this paper, in order to explain the mineral-specific proteins we could assume that oyster secretes one particular calcifying protein to form a specific layer and calcium ions are binding to the COO- groups of amino acid side chains inside these proteins. And to quantitatively investigate the secondary structure of mineral-specific proteins, resolution enhancement techniques, such as Fourier self deconvolution and gaussian curve-fitting techniques were applied. If either the □-helix or □-sheet content in a given system is higher or lower than that of the native form, it could be thought that it is demonstrating the disordering and rearrangement of secondary structure. Also we are to obtain new information on the interaction between calcium ions and mineral-specific proteins. The results show the methods for understanding the principles that govern shell formation, and the insight into the mechanisms by which these shell protein constituents control crystal polymorph in vivo.

2. Experimental Procedure

Shells of Crassostrea Gigas were freshly collected, soaked in 5% NaOH, lightly scrubbed, and dried at room temperature. After completely dried, the shells were finely ground using a mill. The mineral-specific powders of 50gm made into a slurry with distilled water were dialyzed against 10□ of 2% acetic acid in dialysis tubing with a 12,000-14,000 molecular weight cutoff(SIGMA). After complete decalcification (48h), whole extracts were centrifuged at 30,000g for 20min and the soluble matrixes(SM) were separated. The soluble proteins were dialyzed against four changes of double-distilled water at 4℃ over 2 days and then lyophilized. Lyophilized shell proteins, 3□, were dissolved in 1□ of 1mM CaCl2 and 1mM NaCl, and then dialyzed against 3 liters of the same buffer. The samples were lyophilized, and mixed with KBr. The spectra were recorded using a MIDAC GRAMS Fourier transform infrared spectrometer. All spectra were analyzed by second derivatization and resolution enhanced by Fourier self-deconvolution in the amide Ⅰ region prior to Gaussian curve fitting to enable quantification of the secondary structure using the program Origin 5.0. The secondary structure contents were calculated from the area of the each Gaussian bands and their fraction of the total area in the amide Ⅰ spectral region. The band assignment in the amide Ⅰ region to individual component followed in the literature[5]. About 10mg of the soluble matrix of each shell layer was chromatographed on a Mono Q column(Pharmacia, HR 5/5)in 20mM Tris base(pH 8.0), by using a linear 0∼1mM NaCl gradient. The eluent was pooled into several fractions, dialyzed against water and lyophilized. High-performance liquid chromatography was carried out with a Water(models 441, wavelengths 229 and 254nm). The samples were chromatographed by using a reversed-phase c18(5□) column and a 2.5-40% gradient of acetonitrile in sodium acetate, pH 6.5. A steplike gradient composed of 10 equal increments of increasing acetonitrile concentration was used. The peaks were collected and lyophilized. Calcium carbonate was grown by slow diffusion(4days) of (NH4)2CO3 vapor into cell culture dish containing 2□ of 7.5mM CaCl2 in a desiccator. The glass was placed on the bottom of the dish. The effect of proteins from each layer on crystal polymorph was examined by adding them in appropriate amounts to CaCl2 solutions.

3. Results and Discussion

The AmideⅠ region of a infrared spectrum of acetic acid-soluble and HCl-soluble proteins look very similar when the two spectra are compared. After applying Fourier self-deconvolution to the original spectra, several differences could be clearly seen from Fig. 1. The overlapping bands that were summation of several significant components are better resolved in the second derivative spectrum. And analysis of these IR spectra by Gaussian deconvolution allowed the determination of the □-helix and □-sheet contents of the protein obtained by each decalcification methods. The individual components reflect the vibrations of peptide carbonyls involved in different secondary structures. In Table 1, these spectra show the quantitative contributions of each component band to the total AmideⅠcontour of the original spectrum. The main band at the 1640-1630cm-1 frequency range is associated with beta-sheet structure and the band near 1656cm-1 can be assigned to alpha-helix structures. All other bands were assigned to unordered structural elements(beta-turns, random coil and extended chains). In the Gaussian deconvolution of the spectrum of  HCl-soluble proteins, most of the IR absorption is caused by □-helices(1654cm-1), while in acetic acid-soluble proteins, most is caused by □-sheet(1630-1640cm-1). Especially in the case of acetic acid-soluble proteins, it yielded the increased □-sheet content(30.4% vs. 15.8%). These differences indicate that the secondary structures of shell proteins decalcified with either acetic acid or HCl were changing during decalcification. The structural differences between acetic acid-soluble and HCl-soluble proteins could be related to their stability and biological activity.

Under in vitro assays, the formation of protein-bound calcium ions was likely to more easily induce the nucleation of the thermodynamically more stable polymorph calcite. But the mineral-specific proteins obtained from both the calcite-bearing layer and the aragonite-bearing layer induced calcite and aragonite crystals respectively. It means some features of the specific proteins are essential to form the polymorphs. Asp and Glu side chains, which contain carboxylate-rich functional groups, could associate with carbonate biominerals. Through molecular periodicity such as beta-sheets and alpha helices, these protein constituents could provide the longer ranged preorganization for oriented inorganic crystallization. It has been noted that when Asp, Gly, and Ser exist in proteins, more than 50% of them are most likely to be in the □-sheet conformation and in the presence of calcium ions, the Asp-rich proteins adopted the □-sheet conformation. We analyzed  the IR spectra in the amide Ⅰ region of two salt form. The spectral deconvolution reveals that the conformations of proteins of calcium salt form are very different from the one in sodium salt form. In the calcium salt form, the main IR absorption band was a significant increase centered at 1639 by □-sheet. The □-sheet content of the protein of sodium salt form decreases from 30.4% to 26%, while the one of calcium salt form rises from 30.4% to 33.47% with a markedly decreasing the □-helix contents. Compared to the band area assigned to beta-sheet conformations in each proteins, the increased area in beta-sheet conformation in calcium form, along with the decreased area of alpha helices, suggests that calcium ions result in an increase of beta structure at the expense of alpha helical structures. These facts indicate a general structural disordering and rearrangement in the protein in binding with calcium ions.

4.References

1. Lee, D. D., J.Crystal Growth, 102, 262 (1990)

2. Lippmann. F. Sedimentary Carbonate Minerals, Springer, Berlin, 1973

3. Weiner, S., Phil.Trans.R.Soc.Lond, 304B, 425 (1984)

4. Haris, P. I., TIBS,  17, 328 (1992),

5. Dong, A., Biochemistry, 29, 3303 (1990)

Fig. 1.

Table 1