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Ph Dependence of the Coiled-Coil Structure of Keratin Intermediate Filament in Human Hair by 13C NMR Spectroscopy and the Mechanism of Its Disruption

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Ph Dependence of the Coiled-Coil Structure of Keratin Intermediate Filament in Human Hair by 13C NMR Spectroscopy and the Mechanism of Its Disruption Polymer Journal, Vol. 30, No. 2, pp 125-132 (1998) pH Dependence of the Coiled-Coil Structure of Keratin Intermediate Filament in Human Hair by 13C NMR Spectroscopy and the Mechanism of Its Disruption Naoki NISHIKAWA,*·**·t Yoshiaki TANIZAWA,* Shoichi TANAKA,* Yasunobu HoRIGUCHI,* Hitomi MATSUNO,** and Tetsuo AsAKURA**·t * Analytical Research Center, R&D Headquarters, Lion Corp., 13-12 Hirai 7-chome, Edogawa-ku, Tokyo 132, Japan ** Department of Biotechnology, Tokyo University of Agriculture and Technology, Nakacho 2-chome, Koganei, Tokyo 184, Japan (Received July 17, 1997) ABSTRACT: 13C NMR spectra of the low sulfur fraction in S-(carboxymethyl)keratine (SCMKA) which corresponds to the hard-keratin intermediate filament (KIF) in human hair have been observed as a function of pH to clarify its disruption mechanism. The assignment was performed by the amino acid composition, both the distortionless enhancement by polarization transfer (DEPT) spectra and the chemical shift values of the SCMKA sample in 8 M urea solution. 13C NMR spectra at pH 5.0 and 6.0 contain essentially no peaks from the amino acid residues in the rod domain, which is due to its coiled-coil structure having highly restricted mobility. The coiled-coil structure was disrupted between pH 6.0 and 7.0 along with great great increase in peak intensities, which indicated the random coil structure occurred. During this disruption process, single chains with a helical form could not exist because there were no helical peaks in the spectra, or they existed for only a very short time, even if they did. Especially, the peak intensities of the side chains of the negatively charged amino acids, Glu and Asp, and those of the positively charged amino acids, Lys and Arg, increased abruptly at around pH 7.0, and these side chains formed ion-pairing interactions maintaining the coiled-coil structure in the rod domain. The peak intensities of the side chains of Leu and Ile also increased abruptly, indicating that hydrophobic interactions among these side chains in the coiled-coil structure were weakened. When the pH of the SCMKA solution was readjusted to pH 6.0 from 9.6, the 13C NMR spectrum was almost identical to that obtained originally at pH 6.0. Thus, both interactions are considered to contribute to the stability of the coiled-coil structure in the rod domain. KEY WORDS Keratin Intermediate Filament / Low Sulfur Fraction in S-(Carboxymethyl)keratine (SCMKA) / Coiled-Coil Structure / 13C NMR Spectroscopy / Like wool, nail, and horn, human hair is one of hand, the rod domain has four helical segments, in which several mammalian structural components formed from two ex-helix chains associates with each other to form a ex-keratin. 1·2 The histological structure of hair fiber double-stranded coiled-coil rope. This coiled-coil struc­ consists of two components, the cortex and the cuti­ ture was proposed by Crick,21 and Pauling and Corey22 cle. The cortex, comprising 85 to 90% ofhair,3- 5 seems as shown in Figure 1, and the detailed structure has been to be responsible for most physical and mechanical studied by analyses of amino acid sequence, X-ray and properties of hair. This component consists of spindle­ NMR_9,23-2s shaped macrofibrils having two main structures, mi­ The amino acid sequence of the coiled-coil structure crofibril and matrix, which are distinguished by their shows a well-conserved, common heptad repeat of structures and amino acid compositions. 1 - 3·6 - 8 The (a-b-c-d-e-f-g)n (Figure 1). About 75% of all a and d microfibril is a crystalline fibrous protein which is mainly positions is occupied by apolar residues, which stabilize composed of ex-helical proteins with a low content of the arrangement of the two strands by hydrophobic cystine. These structures are aligned along the fiber axis interactions. 29 This regular disposition of the apolar and embedded in an amorphous matrix with a high residues was observed in tropomyosin30 and then content of cystine. Therefore, hair fiber is regarded as recognized also in KIF, 2 · 3 ·9 ·25·26 Recent sequence an oriented fiber in which the crystalline filaments are analyses of KIF have revealed that both positively and aligned, and such a structure must be correlated with the mechanical properties of the keratin fiber. Based on the amino acid sequence and structural homologies, the microfibril of keratin is classified into a member of intermediate filaments (IF) such as vimentine, desumin, glial filaments and neurofila­ ments. 9 - 11 Hence, the microfibril has been recently called a keratin intermediate filament (KIF). All sorts of IF proteins consist of a central rod domain, and N- and C-terminal domains.3·12 - 20 The structure of the N- and Figure 1. Model of coiled-coil structure, drawn schematically.28•33 C-terminal domains has been assumed to be disordered The view which is down the helical axis of a KIF and shows one pair of heptad repeats for each polypeptide chain. Positions a and d are according to amino acid sequence analysis. On the other apolar residues, and an oval lying in the center indicates the hydrophobic interaction area. The oppositely charged amino acid t To whom all correspondence should be addressed. residues in positions e and g form ion pairing (gray bold line). 125 N. NISHIKAWA et al. negatively charged amino acid residues are present in water again, and then these purification procedures positions e and g and distributed periodically in KIF were repeated twice more. The precipitate and super­ molecules. 27 ·28· 31 - 33 This ion-pairing interaction form­ natant fraction containing SCMKA and SCMKB, re­ ing between e and g residues also stabilizes the coiled-coil spectively, was lyophilized. The weight partitions of structure like the hydrophobic interaction. These two SCMKA and SCMKB among SCMK were estimated interactions have been studied by X-ray and NMR for as 65 and 35%, respectively. This partition was similar the GCN4 leucine zipper which forms the coiled-coil to those ofSCMKA and SCMKB obtained from wool. 40 structure. 23 ·24•34 For assignment of the 13C NMR spectra of SCMKA It is generally known that the mechanical properties and analysis of its structure, helix-rich and amorphous­ of hair fiber are changed by chemical cosmetic treatments rich fractions of SCMKA were prepared by enzymic such as permanent-waving, bleaching or dyeing. It is said digestion. Both fractions were prepared by partial hy­ that this decrease is caused by the incomplete re-oxidation drolysis with ix-chymotrypsin from bovine pancreas of the disulfide bond existing in the matrix during these (Wako Pure Chemical Industries, Ltd., Tokyo) as de­ treatment processes. However, the structural change in scribed by Crewther and Dowling.41 The digestion was KIF should be related to the decrease in mechanical terminated by precipitation at pH 4.0. The precipitate properties because of the oriented structure of the hair containing the helix-rich fraction was dissolved at pH fiber. Chemical reagents in alkaline aqueous solutions 9.0 and re-precipitated at pH 4.0. The supernatant are often used for these treatments,3 ·35 - 37 so it is solution obtained by centrifugation contained the important to examine the influence of various pHs on amorphous-rich fraction. These fractions were purified the coiled-coil structure of KIF aligned along the fiber by repeating the precipitation and centrifugation, and axis. Moreover, the influence on the coiled-coil structure then the final solution was lyophilized. The weight is interesting, because it is stabilized by the hydrophobic partitions of the helix-rich and amorphous-rich fractions and ion-pairing interactions formed by several definite in SCMKA were about 30 and 70%, respectively. amino acid residues. In this study, the S-carboxymethyl derivative of kera­ Methods tin protein corresponding to KIF was extracted from The amino acid compositions of cleaned hair, human hair and then analyzed by 13C solution NMR SCMKA, the helix-rich and amorphous-rich fractions spectroscopy in order to clarify the disruption mech­ of SCMKA were determined as follows; 4 mg of each anism of the coiled-coil structure. sample was hydrolyzed in 6 N hydrochloric acid for 24 h at 110°C, dried under nitrogen gas flow and dissolved in EXPERIMENTAL 0.5 ml of citrate buffer (pH 2.2). Filtered solutions were analyzed with an amino acid analyzer (Mitsubishi Kasei Materials Model-AA0I, Tokyo). Experimental errors in the amino Commercial black Asian hair from Staffs Co., Ltd. acid analysis were within 1%. (Tokyo) was used for all experiments. The carboxymethyl The 13C NMR spectra were recorded on a JEOL ix-500 derivative of keratin protein (SCMK) was extracted from NMR spectrometer operating at a 13C frequency of the human hair and then separated into low sulfur 125 MHz at 50°C. Typical NMR conditions for ob­ fraction (SCMKA) and high sulfur fraction (SCMKB), servation are 11000 scans, 34 kHz spectral width and which correspond to microfibril and matrix, respec­ 3.0 s delay between pulses. Distortionless enhancement tively, 38 •39 as follows: The hair bundle was washed with by polarization transfer (DEPT) spectra were also 1 % aqueous solution (w/w) of sodium dodecyl sulfate recorded for the spectral assignment. The concentration (SDS), rinsed thoroughly with deionized water, chopped, of the sample solution was prepared to 5% in 10% (w/w) cleaned by extraction three times with hexane, ethanol, D 2 0, and its pH was adjusted with hydrochloric acid and acetone, and then air-dried at room temperature. and sodium hydroxide. The pH value of each sample This dried hair was wetted with 8 M urea, and reduced solution was again reconfirmed after NMR measure­ by 0.23 M 2-mercaptoethanol overnight at room tem­ ment.
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