Biochemistry 1989, 28, 59 17-5923
5917
Enhanced Sensitivity to Conformation in Various Proteins. Vibrational Circular Dichroism Results? Petr Pancoska,t Sritana C. Yasui, and Timothy A. Keiderling* Department of Chemistry, University of Illinois at Chicago, Box 4348, Chicago, Illinois 60680 Received December 21, 1988; Revised Manuscript Received March 7, 1989
ABSTRACT: Vibrational circular dichroism (VCD) spectra of several globular proteins dissolved in D 2 0 are
presented and compared to conventional UV-CD results. It can be seen that, for the a, P, and a + p categories of Levitt and Chothia [(1976) Nature 261, 5521, VCD evidences much larger band shape variations, including sign alteration, than does UV-CD. A direct parallel is seen between the VCD of the a-helix found in model polypeptides and the amide I’ VCD of myoglobin. Since all structural aspects of the protein contribute to the VCD on a roughly equal footing, a similar correlation of the chymotrypsin amide I’ VCD with that of P-sheet models is not as clear. In addition, the VCD of “random-coil”-type proteins is found to be clearly related to VCD results from “random-coil” polypeptides. Finally, simulations are presented to postulate the expected VCD for protein structures having conformations that lie between the limiting cases discussed here.
Q
ualitative and quantitative aspects of protein secondary structure have been studied with various spectroscopic methods. IR (Byler & Susi, 1986; Yang et al., 1985) and Raman (Parker, 1983; Lippert et al., 1976) spectra are rich in structurally sensitive spectral features, but these spectroscopic data, to date, have been interpreted primarily via frequency shifts and relative intensity changes (Krimm & Bandekar, 1986). Since vibrational spectral properties are mainly determined by the properties of the local oscillators involved in the transitions studied, the secondary structure exerts what might be termed a “second-order”influence upon the spectrum. Even the resolution-enhancing techniques now popular for FTIR studies (Mantsch et al., 1986; Moffatt et al., 1986) do not obviate this fundamental problem. Since it is a chiroptical method, the circular dichroism (CD) spectrum is determined in “first-order” by the molecular conformation. This has led to the wide use of CD measured in the ultraviolet (UV) for empirical studies of protein conformations (Woody, 1977; Brahms & Brahms, 1980; Yang et al., 1986; Johnson, 1985). Numerous studies have followed the initial proposal of Greenfield and Fasman (1969) of using C D curves obtained with model polypeptides to interpret protein data. The prime success of their approach stemmed from the dominance of the contribution from the a-helical components to the near-UV CD spectra as compared to that from other elements of the secondary structure. The inherent qualitative advantage of C D can be exploited quantitatively by development of characteristic and transferable “basis” CD spectra that are typical for individual secondary structures. Fitting linear combinations of them to the experimental data can then yield estimates of the composition of the secondary structure (Chang et al., 1978). Improving on the original idea that used model polypeptides as the source of reference CD data, these basis spectra were subsequently derived from ‘This research was supported by a grant from the National Institutes of Health (GM 30147). The FTIR and UV-CD instruments were purchased with funds provided in part by the National Science Foundation, the National Institutes of Health, and the University of Illinois at Chicago. t Permanent address: Department of Chemical Physics, Charles University, Prague, Czechoslovakia.
0006-2960/89/0428-59 17$01.50/0
protein CD spectra by use of the X-ray-determined secondary structures (Saxena & Wetlaufer, 1971; Chen & Yang, 1971). Subsequent studies avoided the use of “basis” spectra by using an analysis of the protein CD spectra directly (Provencher & Glockner, 1981). Alternatively, such spectra were used for generation of orthogonal basis CD “vectors” which could then be used in a scheme to generate an analysis of the secondary structure of a protein which was not in the original set (Hennessey & Johnson, 1981). Analysis of such results is dependent on the accuracy and stability of the “basis” spectra and on an assumption that there is a linear dependence of the C D intensity on the fraction of a given conformational type in a specific protein. This is equivalent to assuming that segments of different secondary structure do not interact in a way that affects the CD spectrum, which would be true if the UV-CD spectrum were determined by short-range interactions. Since UV-CD spectra are also affected by other factors, the linearity assumption can be a potentially limiting factor in improvements of quantitative conformational analyses. Vibrational circular dichroism (VCD) spectroscopy combines the structural sensitivity of C D with the resolution and local oscillator characteristics of IR and Raman spectroscopies. From the first report of polypeptide VCD (Singh & Keiderling, 1981), application of VCD in the conformational analysis of biopolymers has, in many aspects, paralleled early use of UV-CD in this area. VCD spectra for model (and “standard” in some sense) homopolypeptides in different conformations have been measured for various vibrational modes which are specific to the amide portion of the molecule (Keiderling, 1986) and were shown to correlate to the polypeptide secondary structure (La1 & Nafie, 1982; Sen & Keiderling, 1984; Yasui & Keiderling, 1986a,b; Paterlini et al., 1986; Yasui et al., 1987a,b; Kobrinskaya et al., 1988). Most polypeptide VCD studies have focused on the amide I (predominantly C=O stretch) band, or amide I’ when deuterated, due to its ease of detection in a variety of solvents and intrinsic signal size. The amide A (mainly N-H stretch) is not possible to detect in aqueous or alcohol-based solvents. The amide I1 band (NH bending and C N stretch) often has weak, featureless VCD which lacks distinctive changes for 0 1989 American Chemical Society
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Biochemistry, Vol. 28, No. 14, 1989
different polypeptides or proteins (except for helical conformers; Sen & Keiderling, 1984; Yasui et al., 1986a,b). Before one can develop a quantitative correlation of secondary structure fraction with spectroscopic measurements, it is necessary to demonstrate that such measurements can be reliably made and that they are indeed sensitive to protein structure. That is the purpose of this introductory paper from our laboratory on protein VCD. We herein demonstrate that the amide I’ VCD of proteins in D 2 0 solution can be routinely measured with a good signal-to-noise ratio (S/N) and test some of the experimental constraints on that measurement. From the resulting data for this particular band, a qualitative view emerges that VCD has an enhanced band shape and sign pattern sensitivity, as compared to the variations seen in UV-CD or conventional IR studies, to the structural variations existing among a set of selected proteins representative of typical structural types. Such an advance would not be possible without the improved VCD apparatus design and detector sensitivity available in recent years (Keiderling, 1989). Additionally, we have, in some cases, substantially reduced the artifact problem (Malon & Keiderling, 1988), which required that a VCD base line be obtained with a racemate to eliminate VCD absorption artifacts, a condition unacceptable for biological samples. Both developments have combined to yield higher quality data than were previously available on these biomolecular systems. Our longer term goal is to analyze the quantitative aspects of these data with the presumption that the information content will be complementary to that available with other techniques. As ill be shown in a series of subsequent papers, this demands a statistically more extensive data set and a conceptually different approach than is undertaken here in our initial presentation of the qualitative variations seen in the VCD of proteins. In the next section are detailed our experimental protocols for obtaining protein VCD in the amide I’ and our opinions regarding optimal conditions for measurement of reliable spectra. Following that, selected amide I’ VCD spectra of structurally different proteins are presented which demonstrate the capability of this measurement for differentiation between selected protein structural types using band shapes and a comparison to the more limited variation seen with conventional UV-CD spectra. Finally, results of a calculational exercise are presented which demonstrate the variation in VCD band shape that might be expected for different proportions of secondary structure. EXPERIMENTAL PROCEDURES Materials. Myoglobin (horse skeletal muscle), cytochrome c (horse heart), lysozyme (chicken egg white), chymotrypsin (bovine pancreas), albumin (bovine), and casein (bovine milk) were purchased from Sigma. Accessible hydrogens were deuterium exchanged by dissolving in D 2 0 and lyophilizing twice before final sample preparation. In addition, some solutions of selected unexchanged proteins were freshly prepared in D20, and their VCD spectra were measured to analyze the effects of this prepsration method. Samples were all prepared in the same manner to optimize the VCD S / N ratio in the amide I’ region on our instrument. To 5 mg of dry, exchanged protein in a micro test tube was added 100 p L of D 2 0 (Aldrich) to prepare a final 5% (w/w) solution. An aliquot of the solution was placed between BaF, windows (25” diameter) which were separated by a 25-pm Teflon spacer and held in a standard cell mount. The absorbance of such a protein sample was typically -0.6-0.7 at 1650 cm-l but, if higher or lower, was adjusted to lie within
--
Pancoska et al. this interval by preparation of a new solution prior to VCD measurement. The VCD base line was obtained by using an H-D-exchanged poly(DL-lysine) (Sigma) solution in D 2 0 prepared in the same manner and using the same cell. The concentration of this sample was adjusted empirically to an absorbance of 0.6 at 1650 cm-’. The IR spectrum obtained matched the amide I’ absorption band of the proteins studied sufficiently well for purposes of base-line correction (see Figure 1). For the stock poly(DL-lysine) solutions, a measurable decrease of absorbance was observed within 48 h, so freshly prepared solutions were used for any series of measurements undertaken. Methods. The UIC dispersive and FTIR VCD spectrometers are described in detail elsewhere (Keiderling, 1981, 1989; Malon & Keiderling, 1988); here are mentioned only those details of the procedure that are important or different for protein spectral measurements. Dispersive VCD data were obtained at 1 1 cm-’ resolution (6-mm slit) as the average of four scans with a 10-s time constant. To enhance S/N in the amide I’ region, a cooled high-pass optical filter (CaF,) was mounted in front of two 3 mm X 3 mm MCT detector elements (Infrared Associates) used as an array in order to better match the slit image. Immediately after base-line and sample VCD scans were obtained, single-beam IR transmission spectra were recorded of the sample and of D 2 0 in the same cell under identical conditions. These were subsequently ratioed, and the log was determined to obtain the absorbance spectrum. Final VCD curves were obtained by subtraction of the poly(DL-lysine) base line from the sample spectrum and by calibration with a correction factor derived from a previously obtained scan of a birefringent plate-polarizer assembly (Nafie et al., 1976). For ease of comparison, all spectra have been normalized to a peak absorbance of 1.0 for the amide I’ band. All spectra were subsequently remeasured with our FTIRVCD system (based on a Digilab FTS-60; Malon & Keiderling, 1988) for sake of comparison. These data were obtained at higher resolution (4 cm-I) by averaging 4 blocks of 4096 scans (mirror speed of 0.6 cm/s). Detector and lock-in gains were adjusted to fill the A/D converter with the signal characteristic of the reduced optical band-pass of the D,O solutions. Base-line correction was done in the same way as described above. UV-CD spectra were obtained with a JASCO 5-600 CD instrument over the range of 180-260 nm. Solutions of 0.1-0.2 mg of protein/mL of D,O were placed in a 0.1-cm quartz cell at room temperature, and their spectra were scanned with a 1-nm band-pass at 0.5-s time constant. For all samples under these conditions, the photomultiplier high voltage remained well within the limits required for reliable measurement, even at the lowest wavelength. Base-line correction (previous scan of cell plus solvent) and averaging over three repetitive scans were done by using the standard software. UV-CD data are presented as molar CD (At) on a per unit amide basis. Protein extinction coefficients from Hennessey and Johnson (1 98 1) were used for determination of sample concentration.
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RESULTSAND DISCUSSION Practical Aspects. As a demonstration of the present state of measurement capability with our dispersive VCD instrument in the amide I’ spectral region, examples of averaged base-line and protein sample (albumin) scans are shown in Figure 1. As asserted above, the poly(DL-lysine) base-line distortions are very small compared to the sample VCD. Thus, the base-line correction corresponds primarily to a tilt and shift of the zero line. It should be recalled that the major problem in VCD
Biochemistry, Vol. 28, No. 14, 1989
Protein Vibrational Circular Dichroism
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2: VCD and absorption spectra of albumin ( 5 % solution in D20) in the amide I’ and 11’ regions as recorded on the UIC FTIRVCD instrument [spectral resolution 4 cm-I, average of 4 blocks of 4096 scans, base-line correction with spectrum of pOly(DL-lySine) FIGURE
1750
1650
.
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FREQUENCY (cm‘l) Typical protein amide I’ VCD (top) and IR absorption (bottom) spectra on the UIC dispersive instrument. An uncorrected sample scan (- - -) for albumin ( 5 % solution in D,O) is compared to a base-line scan (-) of a poly(DL-lysine)solution in D 2 0 (see Experimental Procedures for details). The corrected albumin spectrum is shown as a solid line (-). Four scans at 11 cm-’ resolution were accumulated and averaged for both sample and base line. FIGURE 1:
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base-line correction is an artifact signal correlated to the absorption strength that appears to stem from residual birefringence in the optical system. The absorption band of poly(DL-lysine) spectrally overlaps those of all the proteins we have studied and, as noted, was adjusted in intensity to match the protein sample absorbance. Thus, the relative flatness of the base-line scan in Figure 1 leads us to have confidence that our data yield reliable protein VCD band shapes which will then be useful for subsequent detailed analyses. As a measure of the effect of spectral resolution on protein VCD, FTIR-VCD spectra were also obtained for the proteins studied. The albumin VCD shown in Figure 2 is a typical example of such data over the amide I’ and 11’ regions. For all our protein samples, we have found no qualitative differences in the shape of the amide I’ VCD spectra obtained with FTIR-VCD a t 4 cm-’ resolution as compared to those recorded on our dispersive instrument at 10 cm-’ resolution. For albumin, this is borne out by comparison of Figures 1 and 2. Furthermore, the quantitative characteristics of the observed dichroic bands (e.g., frequency positions, relative and also absolute intensities) were comparable to within experimental error. We therefore suppose that no important spectral features are lost when protein VCD spectra are collected at 10 cm-I resolution. From another point of view, this finding allows us roughly to estimate that the discernible VCD features which one can expect to extract from the amide I’ region for protein samples have bandwidths of the order of 10-15 cm-’. Thus, assuming they had a favorable spectral sign pattern, four or five dichroic bands would be theoretically resolvable in the amide I’ region using our instrumental parameters. Deuterium Exchange. The influence of deuteration on the VCD and IR spectra of proteins is an important factor in studying them in D20. Study of the amide I in H 2 0 is not
solution]. possible due to the overlapping HOH deformation bands. We have shown that, for synthetic, regular-sequence polypeptides, the a-helical amide I’ (deuterated) VCD has a significantly different shape than does the amide I (protonated) VCD (Sen & Keiderling, 1984; Yasui & Keiderling, 1986a). On the other hand, we have no indication from our polypeptide data of such effects occurring with other secondary structures (Yasui & Keiderling, 1986a,b). Incomplete deuterati Jn could, in principle, be a problem in obtaining stable, characteristic VCD for proteins. That is one reason we have used carefully controlled sampling conditions for all of the samples studied. To further probe this exchange problem, we have measured VCD of some unexchanged protein samples in the amide I’ region directly after their dissolution in D,O. Due to the large shift of the amide I1 (to 11’) from 1550 to 1450 cm-’, at least for those amides which are exchanged, there is a change in intensity on the lower energy side of the absorption band with deuteration. The differences observed in VCD spectra of deuterated and protonated species are of a quantitative character and vary for individual curve shapes (Le., for different protein types). In general, the qualitative band shape is maintained. In some cases, with bands having alternating signs, features lying between more intense bands may seem to change more significantly due to cancellation effects. These observations lead us to believe that any H-D exchange effects which are important for qualitative modeling of the protein amide I’ VCD spectra primarily involve the fast-exchangeable amide protons (Parker, 197 1). These processes appear to be nearly complete within the first 10 min after the protein sample is dissolved in D 2 0 , Le., before we can observe them. Thus, from a practical point of view, the rigorous H-D exchange procedure we have adopted may not be strictly necessary, if only qualitative analysis of protein amide I’ VCD spectra is desired. On the other hand, for the sake of comparison and for purposes of quantitative analysis of the spectra, careful, systematic H-D exchange is necessary. In addition, these results imply that, if the deuteration process and concomitant lyophilization could induce confor-
5920 Biochemistry, Vol. 28, No. 14, 1989
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Comparison of amide UV-CD from 180 to 260 nm (left) with amide I’ VCD (right) spectra for four different protein samples that represent three types of protein folding patterns (see text). FIGURE 3:
mational changes, such a process could be studied by comparing the VCD of systematically exchanged and unexchanged samples in D 2 0 . The qualitative differences or similarities of the resulting VCD spectra would confirm or deny, respectively, that such a change has taken place. Discrimination Capabilities of VCD. Introduction of a new spectroscopic technique is best justified if it succeeds where either previously used methods have failed or their results are ambiguous or difficult to extract. For VCD, a comparison with its chiroptical predecessor, UV-CD spectroscopy, is natural. Thus, for selected proteins, in Figure 3 we present UV-CD spectra measured over the amide na* and RX*bands (out to 180 nm) for comparison to their corresponding amide I’ VCD counterparts. It becomes immediately obvious that, while the UV-CD spectra have significant magnitude changes and small frequency shifts, in general, all of these proteins give rise to the same three-band C D pattern. Our data are in full agreement with the spectra of others for these proteins (Johnson & Tinoco, 1972; Brahms & Brahms, 1980; Hennessey & Johnson, 1981). On the other hand, the VCD spectra of these same proteins differ substantially in that they change completely in sign pattern and dramatically in shape. As sign variation is the prime advantage of a chiroptical method for empirical analyses, clearly the VCD spectral band shapes are more sensitive to whatever structural differences might exist
among these selected proteins than are the UV-CD spectral band shapes. The protein samples for which spectra are shown in Figure 3 were selected to represent three different types of globular protein structures as first classified by Levitt and Chothia (1976). Their classification scheme refers to the secondary structure type which dominates the ordered segments that fold to create the tertiary structure. Myoglobin belongs to class I (predominantly a-helical, denoted as “all-a” in their paper) and chymotrypsin to class I1 (mainly &sheet, there denoted “all-0”). Cytochrome c and lysozyme both belong to class 111 (having both a and /3 segments that are not intermixed in the protein sequence, there denoted as the “ a + p class”) but have different amounts of 0-structure contribution. It can be seen from Figure 3 that the amide I’ VCD spectra are quite different for the three protein structural types. For class I myoglobin, a negatively biased, positive couplet of bands (extrema at 1666 and 1650 cm-I, crossover point a t 1656 cm-I) dominates the spectrum with an additional weak negative band seen to lower energy (1630 cm-’). Class I1 chymotrypsin is characterized by a negatively biased, negative coupler (extrema at 1652 and 1633 cm-’ and crossover point at 1645 cm-I) with an additional weak positive band lying to higher energy (1698 cm-I). The myoglobin, class I, result is indeed similar in shape to the standard a-helical amide I’ VCD spectrum (Sen & Keiderling, 1984) with the major deviation being in the lowest energy negative band. The class I1 result is only vaguely similar to the model polypeptide antiparallel P-sheet result (Yasui & Keiderling, 1986b) in that it is dominated by a low-energy negative band. Such a difference between model system and the class I1 protein result is reasonable since the 0-sheet fraction is only 34% in chymotrypsin (Henessey & Johnson, 1981). If these two (in some respects, limiting) secondary structural types are combined in proteins of an intermediate structural type, the sum of positive and negative couplets of differently shifted VCD bands would be expected to yield a multiband VCD spectrum. Indeed, lysozyme and cytochrome, the “a p,class 111 examples in Figure 3, have a t least three apparent extrema at 1665 f 5, 1651 f 5, and 1637 f 5 cm-’. It might be noted that these two patterns are different in terms of relative intensities which would have been expected on a qualitative basis from their structural compositions. By contrast, UV-CD curves of the a- and &type proteins (class I and 11) have the same sign for both the long-wavelength (negative) and short-wavelength (positive) transitions. Since the UV-CD band shape is relatively consistent for these selected proteins, the relative differences in intensity are the prime means of qualitative differentiation between the respective protein types. Traditionally, the third primary category of protein secondary structure has been somewhat arbitrarily labeled as “random-coil”. In some studies, this aspect of the conformation has been more accurately labeled as “other”, because, in a globular protein, very little of the structure is disordered or truly “random”. In Figure 4, the C D and VCD spectra of casein are presented as an example of a protein in a “coil” conformation and are there compared to similar results obtained with poly(r,-glutamic acid) in neutral D 2 0 . The UV-CD spectrum of casein (Figure 4a) is indeed qualitatively different from the UV-CD spectra of the class I, 11, and 111proteins shown in Figure 3. It is also qualitatively different from the standard “random-coil” UV-CD used in protein secondary structure analysis which has positive C D in the na* band and a negative maximum a t 195 nm in the
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Biochemistry, Vol, 28, No. 14, 1989 5921
Protein Vibrational Circular Dichroism
Table I: Secondary Structural Compositions Corresponding to VCD Simulationsa myog1obin:casein chym0trypsin:casein myog1obin:chymotrypsin SI:S2 ratio ff 8 0 ff P 0 ff P 0 0 22.0 10.0 34.0 56.0 78.0 0 22.0 1o:o 78.0 0 29.8 9.0 30.6 60.4 71.2 3.4 25.4 9: 1 70.2 23.8 57.6 54.6 0 45.4 7.0 69.2 10.2 32.2 7:3 5.0 17.0 78.0 44.0 17.0 39.0 39.0 0 61.0 5:5 0 76.6 3.0 10.2 86.8 30.4 23.8 45.8 3:7 23.4 3.4 95.6 16.8 30.6 52.6 7.8 0 92.2 1.O 1 :9 0 0 100.0 10.0 34.0 56.0 0 0 100.0 0:lO OValues in the table are percent a-helix, 8-sheet, and other as obtained from a weighted sum of the fraction of each type in each protein. Weights are given by the S1:S2 ratios, and fractional structures are taken from Hennessey and Johnson (1981). UV CD
180
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WAVELENGTH (nm) FREQUENCY (cm”) FIGURE4: UV-CD (a) and amide I’ VCD (b) spectra of casein (5%
solution in DzO) a s compared to UV-CD (c) and amide I’ VCD (d) of poly@-glutamic acid) a s examples of the “coil” conformation. m* region (Greenfield & Fasman, 1969). Panels a and c, respectively, of Figure 4 are a demonstration of the differences that exist between a “random-coil” protein, casein, and a polypeptide standard, poly(L-glutamic acid). The VCD for the “coil” structure as exemplified here by casein has a positive maximum at 1670 cm-’, a crossover at 1655 cm-I, and a negative maximum at 1636 cm-*, with an intensity ratio of 1:3 for the positive to negative bands. This spectrum is qualitatively and quantitatively similar to the VCD measured for poly(L-lysine) (D20, pH 7.1) (Yasui & Keiderling, 1986b; Paterlini et al., 1986), for poly@-glutamic acid) (D20, pH 7.6-11.7) (Dukor & Keiderling, 1989), and for poly(L-tyrosine) (DMSO) (Yasui & Keiderling, 1986a). We have shown that this pattern is consistent with the VCD found for type I1 poly(L-proline) (Kobrinskaya et al., 1988), a left-handed (three residues per turn) helical structure sometimes called the “extended helix” (Tiffany & Krimm, 1968, 1969). In this class of compounds, it is clear that the two chiroptical methods reflect different aspects of the structure. It appears that the VCD results evidence the similarities between the protein and model polypeptide geometries and that the UVC D senses the deviations between them as well. These model “coils” have been postulated to be locally ordered extended helices (Tiffany & Krimm, 1968, 1969) with long-range disorder. We have previously proposed that VCD, on the basis of experimental evidence (Yasui et al., 1986), responds to relatively short-range interactions between subunits in the polypeptide chain. The deviation between VCD and UV-CD
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results seen here is consistent with such a hypothesis. It can be further noted that variability in the qualitative features of a basis spectrum for a given conformation is a violation of the underlying principles used in the simpler methods of quantitative reduction of CD data to give secondary structure information. Such has long been the problem in characterizing the “random-coil”by CD and leads to numerical uncertainty in the results. As such, the apparent qualitative stability of the “coil” VCD spectra, shown in Figure 4, may prove to be an advantage in future quantitative studies. Conformational Variation Effects on VCD. Now we wish to shift our emphasis from the discussion of actual protein CD and VCD data to extrapolate what might be found for the VCD of other proteins. By calculationally combining the experimental VCD curves that we have obtained for myoglobin, chymotrypsin, and casein in different ratios, we can simulate the VCD correspondingto the range of conformations that lie between those exemplified by these three proteins. This, of course, makes a linear additivity assumption analogous to that used in the formulation of CD “basis” spectra. Perhaps less obviously, it assumes that all the spectral features illustrated result from secondary structure considerations. Our data neither confirm nor deny, for VCD, these standard assumptions in CD analyses. Somewhat arbitrarily, these three proteins will here be taken as limiting, but not by any means uniform, examples of structural compositions found for globular proteins in the class I, class 11, and “coil” types. Available X-ray structural data for the proteins can be used to gain some impression as to what protein secondary structural composition could generate VCD spectra intermediate between these limiting cases. Results of our simulations using smoothed limiting spectra (labeled as S1 and S2 in each) are shown in Figures 5-7. The computed VCD curves shown were generated from experimental VCD spectra of the three proteins representing class I, class 11, and “coil” structures by taking their pairwise linear combinations in ratios of 10:0, 9:1, 7:3, 5:5, 3:7, 1:9, and 0:10, respectively. In Table I, the percentages of secondary structural type (a-helical, @-sheet,and “others”, which includes coil, turn, and unclassified conformations) which would compose a protein corresponding to these computed spectra are presented. Values tabulated were derived from the available X-ray determinations of fractional secondary structural types. The VCD curves shown in Figure 5 model the VCD spectral changes expected in going from class I or “a” proteins to ones with increasing contribution for “coil” components. Characteristics of this simulation are an isodichroic point near the zero line, variability of the low-frequency band position, and the possibility of cancellation of bands in the 1660-1680 cm-’ region. Such a family of VCD curves could be expected to be found in an experiment that uses VCD to monitor the gradual conversion of an a-helical sample into a “coil” form. For an example of such a phenomenon, we refer to the mea-
5922 Biochemistry, Vol. 28, No. 14, 1989
Pancoska et al. 1
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Simulated amide I’ VCD as calculated by linear combination of myoglobin (Sl) and casein (S2) VCD spectra. [Sl:S2ratios of 9:l (l), 7:3 (2), 5:5 (3), 3:7 (4), and 1:9 ( 5 ) were used]. See Table I for corresponding percentages of individual secondary structures. FIGURE 5 :
Simulated amide I’ VCD as calculated by linear combination of myoglobin (Sl) and chymotrypsin (S2) VCD spectra. Sl:S2 ratios as in Figure 5 . See Table I for corresponding percentages of individual secondary structures. FIGURE 7:
computed spectra contain a small variable contribution from I the a-helix component.
Simulated amide I’ VCD as calculated by linear combination of chymotrypsin (Sl) and casein (S2) VCD spectra. Sl:S2 ratios as in Figure 5 . See Table I for corresponding percentages of individual secondary structures.
Figure 7 models a variation between our class I and class I1 examples. Again, since the secondary structure of the limiting class I1 model, chymotrypsin, is actually quite mixed, the plotted results have variable contributions from the “coil” form. Hence, the variation modeled covers all three basic secondary structure types. As above, the range of VCD spectra possible in a “pure” a to @ transition could be larger. Due to the qualitative similarity of the “coil” and class I1 amide I’ VCD spectra, this plot has some similarity to Figure 5 ; however, in Figure 7 the isodichroic point is shifted to the positive part of the curve. The resulting VCD spectra, in all cases, have multiple bands, and the position of the low-frequency negative band is less variable than in the Figure 5 simulation between “a” and “coil”. The above calculational exercise may have value in determining qualitative differences in VCD band shapes found in the study of two-state equilibria characteristic of protein structural changes or denaturizations. Additionally, the computed spectra should exemplify most of the amide I’ VCD features that are likely to be found in other proteins.
surement of the VCD of the coil-to-helix transition of poly(L-glutamic acid) as a function of the change of pH (Dukor & Keiderling, 1989). Between the characteristic a-helical (pH 4) and coil (pH 7 ) polypeptide VCD, at pH 5 . 5 , a spectrum was obtained which consisted of a single negative band at 1640 cm-I. This is similar to the computed spectrum 4 in Figure 5. Poly(L-lysine) also has a single-signed VCD spectrum at pH 10.5 (Yasui & Keiderling, 1986b), but in that case, the transition not only involved “coil” and helix but also involved some contribution of the antiparallel P-sheet conformation. Figure 6 models a variation between the class I1 ”@”and “coil” structures. As is clear from comparison of the end-point VCD spectra in this case, the simulation evidences only a small variation in computed VCD as structural composition is varied. The changes predicted would be difficult to detect under real S/N situations. Chymotrypsin, our class I1 model protein, is characterized as having only 34% @-structure. Hence, the range of variation in the VCD measured in monitoring a sheet-coil transition could be substantially larger. Furthermore, since real protein-limiting cases were chosen, these
CONCLUSIONS The VCD spectra we have presented in the amide I’ region for selected protein structural types clearly document that this new chiroptical method exhibits high conformational sensitivity to the details of protein secondary structure, even on a qualitative level. The information which can eventually be derived from these spectra is, in some respects, complementary to that obtainable by UV-CD and other spectroscopic techniques. Intensities of the VCD curves characteristic of different protein secondary structures are comparable. The observed VCD spectrum for a typical protein sample is therefore not dominated by the a-helical contribution as is true in nearUV-CD spectroscopy. Comparability of the contributions from different conformations to the overall protein VCD spectrum should yield a more sensitive evaluation of the proportion of those conformations in future quantitative analyses of these spectra and their dependence on secondary structure. This aspect of VCD is further enhanced by the favorable sign patterns found in the VCD spectra of different protein structural types which, in combination with confor-
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s1
s1 . 1680 1620 FREQUENCY (cm-’)
I 1560
FIGURE 6:
-
Protein Vibrational Circular Dichroism mationally dependent frequency shifts of the relatively narrow IR bands, results in better resolution of the dichroic bands in the amide I’ region as compared to the analogous situation in the UV-CD spectra. The results in this paper demonstrate the possibility of developing a direct and straightforward application of VCD spectroscopy for qualitative characterization of protein structural type in solution. Study of the quantitative aspects of this problem is currently under way in our laboratory.
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