Effect of Temperature on the Conformation of Extended α-Keratin
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552 Volume 55, Number 5, 2001 APPLIED SPECTROSCOPY 0003-7028 / 01 / 5505-0552$2.00 / 0 q 2001 Society for Applied Spectroscopy Effect of Temperature on the Conformation of Extended a -Keratin DONALD J. LYMAN,* JACQUELINE MURRAY-WIJELATH, and MAX FEUGHELMAN Department of Materials Science and Engineering, University of Utah, Salt Lake City, Utah 84112 (D.J.L.); Department of Molecular Biology, The Hope Heart Institute, Seattle, Washington 98122 (J.M.-W.); and Department of Textile Technology, School of Materials Science and Engineering, The University of New South Wales, Sydney, 2052 Australia (M.F.) Attenuated total re ection Fourier transform infrared (ATR/FT- IR) spectra of wet horse hair a -keratin indicate that the confor- mation of extended a -keratin is affected by the water temperature. Extension of the wet horse hair in 21 8 C water gives rise to parallel b -sheet structures, thus suggesting that the original a -helical struc- ture is also parallel. In contrast, extension of the wet horse hair a - keratin in 95 8 C water gives rise to anti-parallel b -sheet structures (consistent with reported literature where X-ray diffraction was used), suggesting that disul de interchange and/or increased chain mobility at elevated temperatures plays a role in altering the sec- ondary structure. Index Headings: ATR/FT-IR; a -Keratin; Conformation. INTRODUCTION Fourier transform infrared (FT-IR) spectroscopy stud- ies on vascular graft healing 1 have shown the appearance formation of extracellular matrix (ECM) proteins within the brin pre-clot used to render polyester vascular grafts impervious to blood as well as forming a hypothrombo- genic luminal surface. A number of these ECM proteins are conformationally similar to the brous proteins. 2 Since the secondary structure of the brous protein horse hair a-keratin has been widely studied by X-ray diffrac- tion, 3,4 it should be useful for obtaining infrared band assignments for the secondary structures of brous pro- teins in the wet state. Since it had been suggested 5 that the conformation of the extended horse hair a-keratin might be due to the elevated temperatures used in the extension process, the extension of the horse hair a-ker- atin was performed at both elevated and room tempera- tures. This paper presents the results of an FT-IR study on these bers using attenuated total re ection (ATR) techniques similar to those used in the vascular healing studies. EXPERIMENTAL Horse hair a-keratin samples with negligible melamine content (donated by Prof. M. Feughelman) were rubbed with ne emery paper to remove any cuticle, rinsed in ether and ethanol, then allowed to dry at room tempera- ture. The horse hair samples were then soaked in deion- ized water for 1 h. One sample was placed on the ATR prism for FT-IR analysis of the wet unstretched hair. A second sample (of about six bers) was stretched ap- proximately 40–50% in 21 8C water, clamped while ex- Received 30 November 2000; accepted 28 December 2000. * Author to whom correspondence should be sent. Present address: P.O. Box 5314, Lacey, WA 98509-5314. tended, then placed on the ATR prism for analysis. A third sample (of about six bers) was stretched approxi- mately 40–50% in 95 8C water, clamped while extended, then placed on the ATR prism for analysis. FT-IR absorption spectra were obtained by using a Di- gilab FTS-40A IR spectrometer with a liquid nitrogen- cooled, mercury-cadmium-telluride (MCT) detector, and a Prism Liquid cell (Harrick Scienti c, Ossining, NY) with a 458 ZnSe crystal (u5 458). Spectra were obtained from 4000 to 700 cm 21 by using 256 scans at a resolution of 2 and Norton–Beer medium apodization. Water sub- traction and other spectral manipulations were carried out by using the GRAMS/386t programs (Galactic Indus- tries, Salem, NH). RESULTS AND DISCUSSION FT-IR spectroscopy has made the study of protein composition and secondary structure a more reasonable task. In addition, the subtractive capabilities of the in- strument in the absorbance mode allow the protein bands of samples, whose spectra are taken in the wet state, to be studied without the interference of overlapping water bands. The large water absorption bands that occur at 3400 and 1640 cm 21 overlap protein absorption bands and must be subtracted. A small band at 2120 cm 21 , due to a combination of water vibrations, 6,7 can be used for the subtraction of water because biochemical materials usually do not absorb at this wavelength. While many vibrational bands give information on pro- tein structure, the amide I band, at 1700 to 1600 cm 21 , is especially sensitive to the secondary structure of proteins. 8 Empirical rules of band positions coupled with a growing number of experimental and theoretical studies 8–16 would appear to allow more accurate band assignments to be made. We have utilized these secondary structural band assignments to determine the effect of temperature (during the stretching process of the wet horse hair) on the re- sulting FT-IR/ATR spectra of the horse hair a-keratin. Wet, Unstretched a -Keratin. The spectrum of the wet, unstretched horse hair and the water-subtracted spec- trum of the wet, unstretched horse hair are shown in Fig. 1A and 1B, respectively. The many sharp peaks in the original spectra, due to water vapor in the sample com- partment, 15 are dif cult to remove experimentally even with extensive purging of the sample chamber. However, smoothing of the spectra, by using the method of Savit- sky and Golay (29 points), 17 reduces these water vapor peaks as noise, and enables one to curve t the data. The smoothed, water-subtracted spectrum of the wet,
Transcript of Effect of Temperature on the Conformation of Extended α-Keratin
552 Volume 55, Number 5, 2001 APPLIED SPECTROSCOPY0003-7028 / 01 / 5505-0552$2.00 / 0q 2001 Society for Applied Spectroscopy
Effect of Temperature on the Conformation ofExtended a -Keratin
DONALD J. LYMAN,* JACQUELINE MURRAY-WIJELATH, andMAX FEUGHELMANDepartment of Materials Science and Engineering, University of Utah, Salt Lake City, Utah 84112 (D.J.L.); Department ofMolecular Biology, The Hope Heart Institute, Seattle, Washington 98122 (J.M.-W.); and Department of Textile Technology, Schoolof Materials Science and Engineering, The University of New South Wales, Sydney, 2052 Australia (M.F.)
Attenuated total re� ection Fourier transform infrared (ATR/FT-IR) spectra of wet horse hair a -keratin indicate that the confor-mation of extended a -keratin is affected by the water temperature.Extension of the wet horse hair in 21 8 C water gives rise to parallelb -sheet structures, thus suggesting that the original a -helical struc-ture is also parallel. In contrast, extension of the wet horse hair a -keratin in 95 8 C water gives rise to anti-parallel b -sheet structures(consistent with reported literature where X-ray diffraction wasused), suggesting that disul� de interchange and/or increased chainmobility at elevated temperatures plays a role in altering the sec-ondary structure.
Index Headings: ATR/FT-IR; a -Keratin; Conformation.
INTRODUCTION
Fourier transform infrared (FT-IR) spectroscopy stud-ies on vascular graft healing1 have shown the appearanceformation of extracellular matrix (ECM) proteins withinthe � brin pre-clot used to render polyester vascular graftsimpervious to blood as well as forming a hypothrombo-genic luminal surface. A number of these ECM proteinsare conformationally similar to the � brous proteins.2
Since the secondary structure of the � brous protein horsehair a-keratin has been widely studied by X-ray diffrac-tion,3,4 it should be useful for obtaining infrared bandassignments for the secondary structures of � brous pro-teins in the wet state. Since it had been suggested 5 thatthe conformation of the extended horse hair a-keratinmight be due to the elevated temperatures used in theextension process, the extension of the horse hair a-ker-atin was performed at both elevated and room tempera-tures. This paper presents the results of an FT-IR studyon these � bers using attenuated total re� ection (ATR)techniques similar to those used in the vascular healingstudies.
EXPERIMENTAL
Horse hair a-keratin samples with negligible melaminecontent (donated by Prof. M. Feughelman) were rubbedwith � ne emery paper to remove any cuticle, rinsed inether and ethanol, then allowed to dry at room tempera-ture. The horse hair samples were then soaked in deion-ized water for 1 h. One sample was placed on the ATRprism for FT-IR analysis of the wet unstretched hair. Asecond sample (of about six � bers) was stretched ap-proximately 40–50% in 21 8C water, clamped while ex-
Received 30 November 2000; accepted 28 December 2000.* Author to whom correspondence should be sent. Present address: P.O.
Box 5314, Lacey, WA 98509-5314.
tended, then placed on the ATR prism for analysis. Athird sample (of about six � bers) was stretched approxi-mately 40–50% in 95 8C water, clamped while extended,then placed on the ATR prism for analysis.
FT-IR absorption spectra were obtained by using a Di-gilab FTS-40A IR spectrometer with a liquid nitrogen-cooled, mercury-cadmium-telluride (MCT) detector, anda Prism Liquid cell (Harrick Scienti� c, Ossining, NY)with a 458 ZnSe crystal (u 5 458). Spectra were obtainedfrom 4000 to 700 cm21 by using 256 scans at a resolutionof 2 and Norton–Beer medium apodization. Water sub-traction and other spectral manipulations were carried outby using the GRAMS/386t programs (Galactic Indus-tries, Salem, NH).
RESULTS AND DISCUSSION
FT-IR spectroscopy has made the study of proteincomposition and secondary structure a more reasonabletask. In addition, the subtractive capabilities of the in-strument in the absorbance mode allow the protein bandsof samples, whose spectra are taken in the wet state, tobe studied without the interference of overlapping waterbands. The large water absorption bands that occur at3400 and 1640 cm21 overlap protein absorption bandsand must be subtracted. A small band at 2120 cm21, dueto a combination of water vibrations,6,7 can be used forthe subtraction of water because biochemical materialsusually do not absorb at this wavelength.
While many vibrational bands give information on pro-tein structure, the amide I band, at 1700 to 1600 cm21, isespecially sensitive to the secondary structure of proteins.8
Empirical rules of band positions coupled with a growingnumber of experimental and theoretical studies8–16 wouldappear to allow more accurate band assignments to bemade. We have utilized these secondary structural bandassignments to determine the effect of temperature (duringthe stretching process of the wet horse hair) on the re-sulting FT-IR/ATR spectra of the horse hair a-keratin.
Wet, Unstretched a -Keratin. The spectrum of thewet, unstretched horse hair and the water-subtracted spec-trum of the wet, unstretched horse hair are shown in Fig.1A and 1B, respectively. The many sharp peaks in theoriginal spectra, due to water vapor in the sample com-partment,15 are dif� cult to remove experimentally evenwith extensive purging of the sample chamber. However,smoothing of the spectra, by using the method of Savit-sky and Golay (29 points),17 reduces these water vaporpeaks as noise, and enables one to curve � t the data.
The smoothed, water-subtracted spectrum of the wet,
APPLIED SPECTROSCOPY 553
FIG. 1. Spectra of the wet, unstretched horse hair a-keratin (A) andwet, unstretched horse hair a-keratin from which water has been sub-tracted (B) [absorbance plotted vs. wavenumber (cm21)]. FIG. 2. Curve-� tted amide I region of wet, unstretched horse hair a-
keratin [absorbance plotted vs. wavenumber (cm21)].
TABLE I. Amide I absorption bands of wet horse hair a -keratin.a
Band (cm21) Band assignment
Unstretched1682165116331618
Disordera-helixb-sheetb-sheet, a-helix
Streatched 40–50% in 218C water16951683164716241611
b-sheet shoulder/b-turnDisorder/b-turnb-sheet parallelb-sheet parallelb-sheet
Stretched 40–50% in 958C water16871666163516141602
b-sheet anti-parallelDisorder/loop, antiparallel b-sheetb-sheet antiparallelb-sheet antiparallelb-sheet
a From curve-� tted bands of ATR/FT-IR spectra.
unstretched horse hair a-keratin was curve � tted in the1750 to 1500 cm21 amide I and II region by using themethod of Marquardt.18 The absorption bands that com-prise the amide I band are shown in Fig. 2 and tabulatedin Table I. The predominant absorption band at 1651cm21 is in the general region considered characteristic ofa-helical structures,8,11,12,16 and thus is assigned to the a-helix structure. The band at 1682 cm21 is in the typicalrange observed for disordered structures. The two ab-sorption bands at 1633 and 1610 cm21 are in the regiontypically seen for b-sheet structures.8,14 The relativelylarge intensity of these two absorptions is undoubtedlydue to the larger molar absorptivity of b-strands, whichare 1.4–1.6 times larger than that of a-helices.19 While itshould be noted that both experimental and theoreticalstudies on myoglobin (which X-ray analysis has shownto possess over 80% a-helix structure and no b-sheetstructure) have shown two small a-helical bands in thisarea,8,14 it is believed that these two bands are related tothe amorphous matrix material.
Thus, the FT-IR/ATR data of the wet, unstretchedhorse hair a-keratin are consistent with a protein that hasapproximately 38% a-helical crystalline regions embed-ded in a disul� de crosslinked amorphous matrix.3
Wet, a -Keratin Stretched at 21 8 C. The smoothed,water-subtracted spectrum of the wet horse hair, stretched40–50% in 21 8C water, was curve � tted as describedabove. The absorption bands that comprise the amide Iregion are shown in Fig. 3 and tabulated in Table I. Ascan be seen, the major a-helix band at 1651 cm21 hasdisappeared and the amide I absorption bands that haveappeared are primarily related to b-sheet structures.8,9,14–16,19
The major absorption band at 1647 cm21 is from parallelb-sheet structures. The other small bands are also related
to parallel b-sheet structures: 1695 cm21 (b-sheet shoul-der); 1683 cm21 (disorder and/or b-turn); and 1624 and1611 cm21 (b-sheet).
Thus, when the wet horse-hair a-keratin � bers arestretched in 21 8C water, a temperature where disul� deinterchange does not occur, the resulting secondary struc-ture would appear to be predominately parallel b-sheetstructures. This observation would suggest that the a-helix structure of the unstretched horse hair a-keratin alsohas a parallel structure.
Wet, a -Keratin Stretched at 95 8 C. The smoothed,water-subtracted spectrum of the wet horse hair, stretched
554 Volume 55, Number 5, 2001
FIG. 3. Curve � tted amide I region of wet horse hair a-keratin stretched45% in 21 8C water [absorbance plotted vs. wavenumber (cm21)].
FIG. 4. Curve � tted amide I region of wet horse hair a-keratin stretched45% in 95 8C water [absorbance plotted vs. wavenumber (cm21)].
approximately 40–50% in 95 8C water, was curve � ttedas described above. The absorption bands that comprisethe amide I region are shown in Fig. 4 and tabulated inTable I. There are major changes in the FT-IR spectrumcompared to that seen when the wet � ber was stretchedat 21 8C. The numerous amide I absorption bands thathave appeared are primarily related to antiparallel b-sheetstructures.8,9–13 The FT-IR/ATR spectrum of the wet horsehair a-keratin stretched in 95 8C water is consistent withthe reported antiparallel structure (from X-ray analyses)for horse hair a-keratin � ber also stretched at elevatedtemperatures.4 These antiparallel structures were retainedwhen the wet stretched horse hair was dried while keptextended (spectrum not shown).
Thus, elevated temperatures play a de� nite role in al-tering the secondary structure of horse hair a-keratin dur-ing the elongation process. This result may be due to thebreakdown (at elevated temperatures) and reformation ofthe disul� de bonds that stabilize the a-helices, or to in-creased chain mobility at these elevated temperatures.3,15
CONCLUSION
The FT-IR/ATR data indicate that extension of the wethorse hair a-keratin at 21 8C gives rise to parallel b-sheetstructures. This information in turn suggests that the a-he-lices of the unstretched horse hair a-keratin are also parallelin structure and not antiparallel, as proposed in the litera-ture.4,20 When the extension of the wet horse hair a-keratin� ber is conducted at elevated temperatures (95 8C), the FT-IR/ATR data indicate that the elongated � ber has an anti-parallel b-sheet structure, consistent with reports in the lit-
erature. Thus, elevated temperatures, which allow the break-down and reformation of disul� de bonds and/or increasedchain mobility, play a prominent role in altering the sec-ondary structure of stretched horse hair a-keratin.
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