- Email: [email protected]
[9] Far (vacuum) ultraviolet circular dichroism
214
CONFORMATION:
OPTICAL SPECTROSCOPY
[8]
rescence excitation spectrum. The excitation spectrum should follow the absorption spectrum of the Y base in the tRNA. Since only Y base absorbs at 335 nm, the excitation spectrum can be scaled using the extinction coefficient at this point. Thus ev is obtained for each wavelength, and the CD spectrum can be calculated. The result of this analysis is shown in Fig. 9. By comparing this spectrum with spectra calculated from various geometries, it is theoretically possible to draw conclusions concerning the conformation around the Y base. Recent progress has shown this to be feasible.'4 Thus FDCD appears to be a powerful new technique for investigating local structure in macromolecules. Acknowledgments I would like to thank 1. Tinoco, Jr., D. S. Moore, M. Maestre, and C. Reich for stimulating discussions. Special thanks are due to 1. Tinoco, Jr., for providing material prior to publication. This work was supported by the Research Corporation and by NIH Grant GM22939-0 I. ,4 D. S. Moore and I. Tinoco, Jr., manuscript in preparation.
[9] F a r ( V a c u u m ) U l t r a v i o l e t C i r c u l a r D i c h r o i s m 1
By EUGENE S. STEVENS Inasmuch as the first measurements of circular dichroism (CD) in the vacuum ultraviolet were made only a few years ago, a brief historical reference ought to be permitted to place that development into a larger perspective. When the general phenomena of rotatory polarization of light and optical activity of chemical substances were elaborated in the first half of the 19th century, it was more often than not the case that experiments would be carried out over a large wavelength region. Biot and Pasteur, for example, regularly measured the wavelength dependence of optical rotation. In the second half of the century, however, experiments were virtually always limited to measurements at the sodium-D line using as a source for that nearly monochromatic radiation the newly invented (1866) bunsen burner. Lowry" in his long review of the subject, "Optical Rotatory Power" written in 1935, describes the invention of the bunsen This work was supported in part by grants from the U.S. Public Health Service (GM22347) and the National Science Foundation (BMS 73-01799). T. M. Lowry, "Optical Rotatory Power." Dover, New York, 1964.
[9]
FAR ULTRAVIOLET CIRCULAR D1CHROISM
2 15
burner as having an inhibiting effect on that branch of optics by making it " a l m o s t too e a s y " to conduct experiments at a single wavelength. Eventually the field r e c o v e r e d and instruments were constructed capable of automatically recording optical rotatory dispersion in the visible and near-ultraviolet regions. Automatically recording CD instruments that operate in the intermediate ultraviolet region (to ~185 nm) were developed only within the last dozen years. The extension of CD measurements into the v a c u u m ultraviolet can be viewed as a natural continuation in the expansion of the CD " w i n d o w . " It should also be noted that the window has also been widened at the other end of the spectral range with the d e v e l o p m e n t of sensitive infrared CD instruments. The fundamental principles of optics which it is necessary to know in order to understand the phenomenon of circular dichroism have been described very often in recent years. A good monograph is that of Velluz, Legrand, and G r o s j e a n ? Tinoco and Cantor have presented a comprehensive and carefully written description of the application of optical rotatory dispersion (ORD) and C D to biopolymers ;~ and Adler et al. have described applications to proteins and p o l y p e p t i d e s ? as have Sears and Beychok," and Timasheff. 7 This chapter is therefore restricted to a description of the special techniques required for m e a s u r e m e n t s of v a c u u m ultraviolet circular dichroism ( V U C D ) and a description of the information that can be obtained f r o m V U C D m e a s u r e m e n t s that are not obtainable from CD m e a s u r e m e n t s at longer wavelengths. Experimental Methods There is at the present time no commercially available instrument tbat allows CD m e a s u r e m e n t s at wavelengths lower than about 185 nm. On the other hand, p r o t o t y p e construction is not prohibitively difficult or expensive. Three instruments have been described in the literature. ~-~" The key c o m p o n e n t in each of these instruments is the quarter-wave retarder. The :~L. Veliuz, M. Legrand, and M. Grosjean, "Optical Circular Dichroism." Academic Pres~, New York, 1965. 1. Tinoco, Jr., and C. R. Cantor, in "Methods of Biochemical Analysis" (D. Giick, ed.), Vol. 18, p. 81. Wiley (lnterscience), New York, 1970. ' A. J. Adler, N. J. Greenfield, and G. D. Fasman, this series Vol. 27, p. 675. " D. W. Sears and S. Beychok, in "Physical Principles and Techniques of Protein Chemistry" (S. J. Leach, ed.), Part C, p. 446, Academic Press, New York, 1973. S. N. Timasheff, in "The Enzymes," 3rd ed. (P. Boyer, ed.), Vol. 2, p. 371. Academic Press, New York, 1970. ~O Schnepp, S. Allen, and E. F. Pearson, Rev. Sci. lnstrum. 41, 1136 119701. "W. C. Johnson, Jr., Rev. Sci. lnstrum. 42, 1283 11971). '" M. A. Young and E. S. Pysh, Macromolecules 6, 790 ( 1973): E. S. t~ysh, Annu. Rev. Biophys. Bioeng. 5, 63 11976).
216
CONFORMATION: OPTICAL SPECTROSCOPY
[9]
Fro. 1. CaF2 quarter-wave retarder. CaF2 crystal is on the left; quartz element is on the right. The retarder shown was obtained from Morvue Electronics, Inc., Tigard, Oregon.
other components of the instruments are conventional2 and include a vacuum ultraviolet monochromator, a hydrogen discharge lamp, a linear polarizer, and a phototube detector. It was the invention of a vacuum ultraviolet quarter-wave retarder that made possible the construction of VUCD instruments. In commercial instruments, ammonium or potassium dihydrogen phosphate (ADE KDP) is used to produce alternately right and left circularly polarized light through the application of the Pockels' effect, but ADP and KDP are not transparent far into the vacuum ultraviolet region. On the other hand, materials that transmit vacuum ultraviolet radiation, such as LiF, CaFz, and MgF2, do not have the crystal symmetry required for exhibiting the electrooptic effect. These materials, however, do exhibit the piezooptic effect. If a piezoelectric transducer, such as quartz, is coupled to a piezooptic device, so as to combine the (inverse) piezoelectric and piezooptic effects, the net result is an electropiezooptic modulator. Kemp ~1 has described just such a modulator. The modulators have been commercially available through Morvue Electronics Systems, Inc., of Tigard, Oregon. A 50 kHz modulation is applied, and window materials of either fused silica or CaF2 are used (Figs. 1 and 2). Instruments of this design require calibration, as do commercial in~ J. C. Kemp, J. Opt. Soc. Am. 59, 950 (1969).
[9]
FAR ULTRAVIOLET CIRCULAR D1CHROISM
217
FIG. 2. Top view of sample chamber in vacuum ultraviolet circular dichroism instrument described by M. A. Young and E. S. Stevens [Macromolecules 6,790(1973)]. Quarter-wave retarder is shown at top center. Photomultiplier housing is shown at bottom center, below V-block sample holder.
struments. Velluz, Legrand, and Grosjean :~have discussed the factors that determine CD instrument sensitivity and precision. The instruments described in the literature have a sensitivity, in units of optical density, of approximately one part in 105 at a 1.6-nm spectral width. The number of prototypes will probably increase, and it can be expected that there may be some modification o f design, aimed at decreasing the cost of construction, and increasing convenience and sensitivity. Of prime importance is the requirement that sufficient light reach the photodetector to ensure that the detected signal indicates the actual dichroism. Optimum signal : noise ratios occur when the absorbance of the sample is near unity, and artifacts can occur if the absorbance is as high as 2.0. Since samples are likely to absorb more strongly in the vacuum ultraviolet than at higher wavelengths, very small path lengths are required. Commercial ceils are available with path lengths of 100 txm (0. I ram) and 50 /xm. In our laboratory, spectra of aqueous solutions can typically be obtained in a commercial 50-/xm cell to 175 nm. Cells with shorter pathlength can be constructed. One design which has been used in our laboratory consists of two CaF., disks separated by aluminum foil spacers. The thin layer of solution is protected from evaporation by sealing the edges with O tings under light pressure. With such a cell we have
2 18
CONFORMATION;
OPTICAL SPECTROSCOPY
[9]
reported spectra of trifluoroethanol solutions to 162 nm. Johnson and Tinoco ~2reported that 160 nm can be reached with hexafiuoroisopropanol. Film spectra can be studied much further into the vacuum ultraviolet. We find that the wettability of the window material and the consequent overall morphological characteristics of the film determine the wavelength penetration limit. It is generally easier to prepare an adequate film from nonaqueous solutions than from aqueous solutions. An orientation of large molecules develops very easily in thin samples; this introduces the danger of a linear dichroism signal being superimposed on the CD signal. A simple test for the absence of linear dichroism consists of rotating the sample about the optical axis. Any net orientation within the plane perpendicular to the light beam will result in an orientation dependence of the signal. The test does not reveal orientation along the direction parallel to the light beam, but usually such orientation is unlikely.
Applications The VUCD of all regular peptide structures has now been reported: the alpha helix, l°'r' polyproline I, ~3 polyproline II, 1:~ the antiparallel pleated sheet, ~4'~5 and the parallel pleated sheet. 14'~ Figure 3 shows the VUCD spectrum of alpha helical poly-y-methyl-L-glutamate in hexafluoroisopropanol reported by Johnson and Tinoco. r-' It is virtually identical to the spectrum of alpha helical poly-L-alanine films cast from trifluoroethanol, z° which indicates that not only are the low-energy CD features insensitive to side chain but that the high-energy features are as well. It is also interesting that the CD of the solution and film are the same over the entire overlapping range. The film spectrum shows a positive band near 140 nm below which the dichroism falls back to the base line near 135 nm. Figure 4 shows the VUCD of poly-L-proline films cast from trifluoroethanol at successively longer times following dissolution. ~:~The spectrum obtained first (3 rain) represents poly-L-proline I; the spectrum obtained after 52 hr is that of poly-L-proline II. In the region where these spectra overlap solution CD spectra, the shapes are nearly identical. Figure 5 displays the VUCD film spectra of the oligopeptide series ~ W. C. Johnson, Jr., and I. Tinoco, Jr., J. Am. Chem. Soc. 94, 4389 (1972). H M. A. Young and E. S. Pysh, J. A m . Chem. Soc. 97, 5100 (1975). ,4 j. S. Balcerski, E. S. Pysh, G. M. Bonora, and C. Toniolo, J. Amer. Chem. Soc. 98, 3470 (1976). ~:' M. Kelly, E. S. Pysh, G. M. Bonora, and C. Toniolo, J. Amer. Chem. Soc. 99, 3264 (1977).
[9]
219
FAR ULTRAVIOLET CIRCULAR DICHROISM I
i
i
i
i
i
I
!
1
,
+20
~
+tO
o~
,
8 To
-t0
t
~40
I
I
~60
q
180
I
I
k (nrn)
200
~
220
i
240
FIG. 3. Circular dichroism (CD) and absorption (abs.) of poly-7-methyI-L-glutamate in hexafluoro-2-propanol. Reprinted with permission from W. C. Johnson, Jr. and I. Tinoco, Jr., J. A m . Chem. Soc. 94, 4389(1972). Copyright by the American Chemical Society.
i
T
r
T
q
~
p
i
I
i
F
i
.08
.04
E ~_j-.04 hi
150
h
i
__
200 WAVELENGTH (nm)
_
250
_
FIG. 4. Circular dichroism of poly-L-proline films cast from trifluoroethanol: (I) 3 min, polyproline 1, (2) 88 min, (3) 179 min, (4) 291 min, (5) 52 hr, polyproline 11. Reprinted with permission from M. A. Young and E. S. Pysh, J. A m . Chem. Soc. 97, 5100 (1975). Copyright by the American Chemical Society.
220
[9]
C O N F O R M A T I O N : OPTICAL SPECTROSCOPY
I
I
I
I
I
I
I
f
I
I
_
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~~
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-
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140
160
180
,
200
-
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220
,
$)40
Wavelength (nm) FIG. 5. Circular dichroism of BOC(L-AIa),OMe, n = 2-7. Reprinted with permission from J. S. Balcerski, E. S. Pysh, G. M. Bonora, and C. Toniolo,J. A m . C h e m . Soc. 98, 3470 (1976). Copyright by the American Chemical Society. BOC(L-Ala),OMe where n-2-7, obtained by Balcerski e t al. ~ The figure shows that the beta conformation predominates in the solid state for all oligomers except the dimer. The authors also reported data for the series BOC(L-Val),OMe. ~ Although the spectra of the alanine heptamer and the valine h e p t a m e r are similar a b o v e 195 rim, there are differences in the 175-190-nm region. Alanine displays a shoulder in the positive band near 185 nm which is absent in the spectrum of the valine heptamer. This difference results in a difference in crossover between alanine (178 nm) and valine ( 192 nm). This difference is the same as the difference between the C D of an antiparallel and that of a parallel sheet expected on the basis of calculations. "~-'° On those grounds the authors cite the likelihood that the alanine heptamer is predominantly in the antiparallel beta sheet and that the valine h e p t a m e r is largely in the parallel sheet conformation. Furthermore, the composite V U C D spectrum of the alanine and valine spectra (Fig. 6) has features identical to the spectrum experimentally measured for BOC(L-Nva)7OMe, and the conformation of that beta sheet is described as containing a mixture of parallel and antiparallel chains. Kelly e t al. ~:' reported a V U C D spectrum for BOC(L-Leu)¢OMe which is ~'; E. ': E. J~ K. '" V. ~" R.
S. Pysh, Proc. Natl. Acad. Sci. U.S.A. 56, 825 (1966). S. Pysh, J. Chem. Phys. 52, 4723 (1970). Rosenheck and B. Sommer, J. Chem. Phys. 46, 532 (1967). Madison and J. Schellman, Biopolymers 11, 1041 (1972). Woody, Biopolymetw 8, 669 (1969).
[9]
221
FAR ULTRAVIOLET CIRCULAR DICHROISM I
0.25
~'~
I
1
I
I
i
I
I
I
(b) i~
030
020
I
(a)
(~15 010
~
0.05 0 - 005
140
Wavelength (nm) FIG. 6. (a) Circular dichroism of BOC(L-AIa)7OMe; (b) circular dichroism of BOC(LVal)rOMe, (c) composite circular dichroism calculated by combining (a) and (b) in equal weights. Reprinted with permission from J. S. Balcerski, E. S. Pysh, G. M, Bonora, and C. Toniolo, J. Am. Chem. Soc. 98, 3470 (1976). Copyright by the American Chemical Society.
similar to that of BOC(L-Nva)rOMe, and its conformation has been assigned also to a mixture of parallel and antiparallei chains. This differentiation between parallel and antiparallel oriented peptide chains is the first major application of VUCD to the study of peptides and proteins. No VUCD protein spectra have yet been reported. Once they become available it will be important to establish whether backbone conformation can be extracted more reliably if experimental data over a larger wavelength range are used. That is, do the differences in the VUCD spectra of the various regular geometries (Figs. 3-6) in the 175-190-nm region provide enough additional information to improve the ability to decompose a protein CD spectrum into additive contributions from a small number of conformational elements? The VUCD spectrum of a standard "disordered" peptide chain would, of course, also be required. Since lipids and saccharides generally absorb only in the vacuum ultraviolet region, the technique will undoubtedly be applied to the study of lipoproteins and mucoproteins.
CONFORMATION:
OPTICAL SPECTROSCOPY
[8]
rescence excitation spectrum. The excitation spectrum should follow the absorption spectrum of the Y base in the tRNA. Since only Y base absorbs at 335 nm, the excitation spectrum can be scaled using the extinction coefficient at this point. Thus ev is obtained for each wavelength, and the CD spectrum can be calculated. The result of this analysis is shown in Fig. 9. By comparing this spectrum with spectra calculated from various geometries, it is theoretically possible to draw conclusions concerning the conformation around the Y base. Recent progress has shown this to be feasible.'4 Thus FDCD appears to be a powerful new technique for investigating local structure in macromolecules. Acknowledgments I would like to thank 1. Tinoco, Jr., D. S. Moore, M. Maestre, and C. Reich for stimulating discussions. Special thanks are due to 1. Tinoco, Jr., for providing material prior to publication. This work was supported by the Research Corporation and by NIH Grant GM22939-0 I. ,4 D. S. Moore and I. Tinoco, Jr., manuscript in preparation.
[9] F a r ( V a c u u m ) U l t r a v i o l e t C i r c u l a r D i c h r o i s m 1
By EUGENE S. STEVENS Inasmuch as the first measurements of circular dichroism (CD) in the vacuum ultraviolet were made only a few years ago, a brief historical reference ought to be permitted to place that development into a larger perspective. When the general phenomena of rotatory polarization of light and optical activity of chemical substances were elaborated in the first half of the 19th century, it was more often than not the case that experiments would be carried out over a large wavelength region. Biot and Pasteur, for example, regularly measured the wavelength dependence of optical rotation. In the second half of the century, however, experiments were virtually always limited to measurements at the sodium-D line using as a source for that nearly monochromatic radiation the newly invented (1866) bunsen burner. Lowry" in his long review of the subject, "Optical Rotatory Power" written in 1935, describes the invention of the bunsen This work was supported in part by grants from the U.S. Public Health Service (GM22347) and the National Science Foundation (BMS 73-01799). T. M. Lowry, "Optical Rotatory Power." Dover, New York, 1964.
[9]
FAR ULTRAVIOLET CIRCULAR D1CHROISM
2 15
burner as having an inhibiting effect on that branch of optics by making it " a l m o s t too e a s y " to conduct experiments at a single wavelength. Eventually the field r e c o v e r e d and instruments were constructed capable of automatically recording optical rotatory dispersion in the visible and near-ultraviolet regions. Automatically recording CD instruments that operate in the intermediate ultraviolet region (to ~185 nm) were developed only within the last dozen years. The extension of CD measurements into the v a c u u m ultraviolet can be viewed as a natural continuation in the expansion of the CD " w i n d o w . " It should also be noted that the window has also been widened at the other end of the spectral range with the d e v e l o p m e n t of sensitive infrared CD instruments. The fundamental principles of optics which it is necessary to know in order to understand the phenomenon of circular dichroism have been described very often in recent years. A good monograph is that of Velluz, Legrand, and G r o s j e a n ? Tinoco and Cantor have presented a comprehensive and carefully written description of the application of optical rotatory dispersion (ORD) and C D to biopolymers ;~ and Adler et al. have described applications to proteins and p o l y p e p t i d e s ? as have Sears and Beychok," and Timasheff. 7 This chapter is therefore restricted to a description of the special techniques required for m e a s u r e m e n t s of v a c u u m ultraviolet circular dichroism ( V U C D ) and a description of the information that can be obtained f r o m V U C D m e a s u r e m e n t s that are not obtainable from CD m e a s u r e m e n t s at longer wavelengths. Experimental Methods There is at the present time no commercially available instrument tbat allows CD m e a s u r e m e n t s at wavelengths lower than about 185 nm. On the other hand, p r o t o t y p e construction is not prohibitively difficult or expensive. Three instruments have been described in the literature. ~-~" The key c o m p o n e n t in each of these instruments is the quarter-wave retarder. The :~L. Veliuz, M. Legrand, and M. Grosjean, "Optical Circular Dichroism." Academic Pres~, New York, 1965. 1. Tinoco, Jr., and C. R. Cantor, in "Methods of Biochemical Analysis" (D. Giick, ed.), Vol. 18, p. 81. Wiley (lnterscience), New York, 1970. ' A. J. Adler, N. J. Greenfield, and G. D. Fasman, this series Vol. 27, p. 675. " D. W. Sears and S. Beychok, in "Physical Principles and Techniques of Protein Chemistry" (S. J. Leach, ed.), Part C, p. 446, Academic Press, New York, 1973. S. N. Timasheff, in "The Enzymes," 3rd ed. (P. Boyer, ed.), Vol. 2, p. 371. Academic Press, New York, 1970. ~O Schnepp, S. Allen, and E. F. Pearson, Rev. Sci. lnstrum. 41, 1136 119701. "W. C. Johnson, Jr., Rev. Sci. lnstrum. 42, 1283 11971). '" M. A. Young and E. S. Pysh, Macromolecules 6, 790 ( 1973): E. S. t~ysh, Annu. Rev. Biophys. Bioeng. 5, 63 11976).
216
CONFORMATION: OPTICAL SPECTROSCOPY
[9]
Fro. 1. CaF2 quarter-wave retarder. CaF2 crystal is on the left; quartz element is on the right. The retarder shown was obtained from Morvue Electronics, Inc., Tigard, Oregon.
other components of the instruments are conventional2 and include a vacuum ultraviolet monochromator, a hydrogen discharge lamp, a linear polarizer, and a phototube detector. It was the invention of a vacuum ultraviolet quarter-wave retarder that made possible the construction of VUCD instruments. In commercial instruments, ammonium or potassium dihydrogen phosphate (ADE KDP) is used to produce alternately right and left circularly polarized light through the application of the Pockels' effect, but ADP and KDP are not transparent far into the vacuum ultraviolet region. On the other hand, materials that transmit vacuum ultraviolet radiation, such as LiF, CaFz, and MgF2, do not have the crystal symmetry required for exhibiting the electrooptic effect. These materials, however, do exhibit the piezooptic effect. If a piezoelectric transducer, such as quartz, is coupled to a piezooptic device, so as to combine the (inverse) piezoelectric and piezooptic effects, the net result is an electropiezooptic modulator. Kemp ~1 has described just such a modulator. The modulators have been commercially available through Morvue Electronics Systems, Inc., of Tigard, Oregon. A 50 kHz modulation is applied, and window materials of either fused silica or CaF2 are used (Figs. 1 and 2). Instruments of this design require calibration, as do commercial in~ J. C. Kemp, J. Opt. Soc. Am. 59, 950 (1969).
[9]
FAR ULTRAVIOLET CIRCULAR D1CHROISM
217
FIG. 2. Top view of sample chamber in vacuum ultraviolet circular dichroism instrument described by M. A. Young and E. S. Stevens [Macromolecules 6,790(1973)]. Quarter-wave retarder is shown at top center. Photomultiplier housing is shown at bottom center, below V-block sample holder.
struments. Velluz, Legrand, and Grosjean :~have discussed the factors that determine CD instrument sensitivity and precision. The instruments described in the literature have a sensitivity, in units of optical density, of approximately one part in 105 at a 1.6-nm spectral width. The number of prototypes will probably increase, and it can be expected that there may be some modification o f design, aimed at decreasing the cost of construction, and increasing convenience and sensitivity. Of prime importance is the requirement that sufficient light reach the photodetector to ensure that the detected signal indicates the actual dichroism. Optimum signal : noise ratios occur when the absorbance of the sample is near unity, and artifacts can occur if the absorbance is as high as 2.0. Since samples are likely to absorb more strongly in the vacuum ultraviolet than at higher wavelengths, very small path lengths are required. Commercial ceils are available with path lengths of 100 txm (0. I ram) and 50 /xm. In our laboratory, spectra of aqueous solutions can typically be obtained in a commercial 50-/xm cell to 175 nm. Cells with shorter pathlength can be constructed. One design which has been used in our laboratory consists of two CaF., disks separated by aluminum foil spacers. The thin layer of solution is protected from evaporation by sealing the edges with O tings under light pressure. With such a cell we have
2 18
CONFORMATION;
OPTICAL SPECTROSCOPY
[9]
reported spectra of trifluoroethanol solutions to 162 nm. Johnson and Tinoco ~2reported that 160 nm can be reached with hexafiuoroisopropanol. Film spectra can be studied much further into the vacuum ultraviolet. We find that the wettability of the window material and the consequent overall morphological characteristics of the film determine the wavelength penetration limit. It is generally easier to prepare an adequate film from nonaqueous solutions than from aqueous solutions. An orientation of large molecules develops very easily in thin samples; this introduces the danger of a linear dichroism signal being superimposed on the CD signal. A simple test for the absence of linear dichroism consists of rotating the sample about the optical axis. Any net orientation within the plane perpendicular to the light beam will result in an orientation dependence of the signal. The test does not reveal orientation along the direction parallel to the light beam, but usually such orientation is unlikely.
Applications The VUCD of all regular peptide structures has now been reported: the alpha helix, l°'r' polyproline I, ~3 polyproline II, 1:~ the antiparallel pleated sheet, ~4'~5 and the parallel pleated sheet. 14'~ Figure 3 shows the VUCD spectrum of alpha helical poly-y-methyl-L-glutamate in hexafluoroisopropanol reported by Johnson and Tinoco. r-' It is virtually identical to the spectrum of alpha helical poly-L-alanine films cast from trifluoroethanol, z° which indicates that not only are the low-energy CD features insensitive to side chain but that the high-energy features are as well. It is also interesting that the CD of the solution and film are the same over the entire overlapping range. The film spectrum shows a positive band near 140 nm below which the dichroism falls back to the base line near 135 nm. Figure 4 shows the VUCD of poly-L-proline films cast from trifluoroethanol at successively longer times following dissolution. ~:~The spectrum obtained first (3 rain) represents poly-L-proline I; the spectrum obtained after 52 hr is that of poly-L-proline II. In the region where these spectra overlap solution CD spectra, the shapes are nearly identical. Figure 5 displays the VUCD film spectra of the oligopeptide series ~ W. C. Johnson, Jr., and I. Tinoco, Jr., J. Am. Chem. Soc. 94, 4389 (1972). H M. A. Young and E. S. Pysh, J. A m . Chem. Soc. 97, 5100 (1975). ,4 j. S. Balcerski, E. S. Pysh, G. M. Bonora, and C. Toniolo, J. Amer. Chem. Soc. 98, 3470 (1976). ~:' M. Kelly, E. S. Pysh, G. M. Bonora, and C. Toniolo, J. Amer. Chem. Soc. 99, 3264 (1977).
[9]
219
FAR ULTRAVIOLET CIRCULAR DICHROISM I
i
i
i
i
i
I
!
1
,
+20
~
+tO
o~
,
8 To
-t0
t
~40
I
I
~60
q
180
I
I
k (nrn)
200
~
220
i
240
FIG. 3. Circular dichroism (CD) and absorption (abs.) of poly-7-methyI-L-glutamate in hexafluoro-2-propanol. Reprinted with permission from W. C. Johnson, Jr. and I. Tinoco, Jr., J. A m . Chem. Soc. 94, 4389(1972). Copyright by the American Chemical Society.
i
T
r
T
q
~
p
i
I
i
F
i
.08
.04
E ~_j-.04 hi
150
h
i
__
200 WAVELENGTH (nm)
_
250
_
FIG. 4. Circular dichroism of poly-L-proline films cast from trifluoroethanol: (I) 3 min, polyproline 1, (2) 88 min, (3) 179 min, (4) 291 min, (5) 52 hr, polyproline 11. Reprinted with permission from M. A. Young and E. S. Pysh, J. A m . Chem. Soc. 97, 5100 (1975). Copyright by the American Chemical Society.
220
[9]
C O N F O R M A T I O N : OPTICAL SPECTROSCOPY
I
I
I
I
I
I
I
f
I
I
_
0.I0 -
~~
//II
a
-
/1 \/
AT/,
140
160
180
,
200
-
\\
,
220
,
$)40
Wavelength (nm) FIG. 5. Circular dichroism of BOC(L-AIa),OMe, n = 2-7. Reprinted with permission from J. S. Balcerski, E. S. Pysh, G. M. Bonora, and C. Toniolo,J. A m . C h e m . Soc. 98, 3470 (1976). Copyright by the American Chemical Society. BOC(L-Ala),OMe where n-2-7, obtained by Balcerski e t al. ~ The figure shows that the beta conformation predominates in the solid state for all oligomers except the dimer. The authors also reported data for the series BOC(L-Val),OMe. ~ Although the spectra of the alanine heptamer and the valine h e p t a m e r are similar a b o v e 195 rim, there are differences in the 175-190-nm region. Alanine displays a shoulder in the positive band near 185 nm which is absent in the spectrum of the valine heptamer. This difference results in a difference in crossover between alanine (178 nm) and valine ( 192 nm). This difference is the same as the difference between the C D of an antiparallel and that of a parallel sheet expected on the basis of calculations. "~-'° On those grounds the authors cite the likelihood that the alanine heptamer is predominantly in the antiparallel beta sheet and that the valine h e p t a m e r is largely in the parallel sheet conformation. Furthermore, the composite V U C D spectrum of the alanine and valine spectra (Fig. 6) has features identical to the spectrum experimentally measured for BOC(L-Nva)7OMe, and the conformation of that beta sheet is described as containing a mixture of parallel and antiparallel chains. Kelly e t al. ~:' reported a V U C D spectrum for BOC(L-Leu)¢OMe which is ~'; E. ': E. J~ K. '" V. ~" R.
S. Pysh, Proc. Natl. Acad. Sci. U.S.A. 56, 825 (1966). S. Pysh, J. Chem. Phys. 52, 4723 (1970). Rosenheck and B. Sommer, J. Chem. Phys. 46, 532 (1967). Madison and J. Schellman, Biopolymers 11, 1041 (1972). Woody, Biopolymetw 8, 669 (1969).
[9]
221
FAR ULTRAVIOLET CIRCULAR DICHROISM I
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Wavelength (nm) FIG. 6. (a) Circular dichroism of BOC(L-AIa)7OMe; (b) circular dichroism of BOC(LVal)rOMe, (c) composite circular dichroism calculated by combining (a) and (b) in equal weights. Reprinted with permission from J. S. Balcerski, E. S. Pysh, G. M, Bonora, and C. Toniolo, J. Am. Chem. Soc. 98, 3470 (1976). Copyright by the American Chemical Society.
similar to that of BOC(L-Nva)rOMe, and its conformation has been assigned also to a mixture of parallel and antiparallei chains. This differentiation between parallel and antiparallel oriented peptide chains is the first major application of VUCD to the study of peptides and proteins. No VUCD protein spectra have yet been reported. Once they become available it will be important to establish whether backbone conformation can be extracted more reliably if experimental data over a larger wavelength range are used. That is, do the differences in the VUCD spectra of the various regular geometries (Figs. 3-6) in the 175-190-nm region provide enough additional information to improve the ability to decompose a protein CD spectrum into additive contributions from a small number of conformational elements? The VUCD spectrum of a standard "disordered" peptide chain would, of course, also be required. Since lipids and saccharides generally absorb only in the vacuum ultraviolet region, the technique will undoubtedly be applied to the study of lipoproteins and mucoproteins.