J. Phys. Chem. B 2001, 105, 7857-7864
7857
Temperature Influence on the Secondary Structure of Avidin and Avidin-Biotin Complex: A Vibrational Circular Dichroism Study Feng Wang and Prasad L. Polavarapu* Department of Chemistry, Vanderbilt UniVersity, NashVille, Tennessee 37235 ReceiVed: February 2, 2001; In Final Form: June 6, 2001
Vibrational absorption and vibrational circular dichroism spectra of avidin with and without d-biotin in phosphate buffer were recorded in the amide I′ (1800-1600 cm-1) region as a function of temperature. The differences in the unfolding pathway of avidin in the presence and absence of biotin were examined using the curve-fitting results of absorption spectra, and the variable-temperature absorption and VCD spectra. This study reveals, contrary to previous spectroscopic studies, but in agreement with X-ray structural studies, that avidin contains β-sheet structures with turns and bends, but does not contain R-helical structure. Also a cooperative structural transition leading to formation of aggregated antiparallel β-strands, with increasing temperature, has been inferred. In avidin-biotin complex, however, some reversible unfolding of β-sheet structure is noted but a cooperative structural transition has not been noted with increase in temperature. Two-dimensional (2D)-VCD correlation spectroscopy has also been used to analyze the sequence of events in structural unfolding of avidin.
Introduction Avidin, a basic tetrameric glycoprotein (4 × 128 amino acid residues, Mr ) 62 400) isolated from egg-white, is being used extensively in a vast number of areas1 for its unusually strong interaction with biotin (vitamin H). Each avidin can bind up to four molecules of biotin with high affinity2 (dissociation constant KD ) 6 × 10-16 M, which is among the strongest known protein-ligand interactions). Avidin and its complex with biotin have been studied using vibrational spectroscopy,3 crystallographic analysis,4a,b and electronic circular dichroism (ECD).4c Honzatko and Williams3a concluded that the secondary structure of avidin consists of a higher proportion of β-sheet structure (55%) than R-helical structure (10%) using Raman spectroscopy. Barbucci et al.3b suggested that the secondary structure of avidin consists mainly of R-helices and β-sheets by FT-IR/ATR. Another FTIR spectroscopic study3c suggested a secondary structural content composed of approximately 66% β-sheet and the extended structures, with the remainder being attributed to disordered structure and β-turns (without R-helices). Raman spectroscopic results3d,e also indicated some conformational changes of avidin upon its binding with biotin, such as a decrease in the percentage of β-sheet and an increase in the percentage of R-helix. The tertiary structure3d of avidin also changes slightly as a result of the biotin binding. From the ECD data4c it was concluded that there is little or no R-helical structure present in avidin, and the crystallographic analysis4a,b has shown no evidence for R-helical structure in the crystal structure. Each of the four monomers of avidin molecule is organized in an orthogonal eight-stranded antiparallel β-barrel with the strands connected by extended loops. With both hydrophobic and polar residues for recognition of vitamin, the binding site is located in a deep pocket at the center of the β-barrel. All the intramolecular hydrogen bonds present in the biotin-binding pocket yield a well-defined rigid structure for binding to the ureidic ring of biotin. * Corresponding author.
Variable-temperature investigation of protein has been used to determine the effect of temperature on the molecular structural stability of avidin.5-7 Donovan and Ross5 reported that biotindepleted avidin and biotin-avidin complex denatured at 85 °C and 132 °C, respectively, in the PH range of 7 to 9. Moreover the avidin-biotin complex is stable in 9 M urea, in the PH range of 2 to 13, and is not denatured in 7 M guanidinium hydrochloride.6 Ismoyo et al.7 studied the effect of heating on the secondary structure by 2D-IR cross-correlation maps generated from Fourier self-deconvolved spectra. The disruption of β-sheet at 85 °C is observed for biotin-depleted avidin. Another FTIR study3c showed that the structural changes that occur in thermal denaturation of avidin take place cooperatively at ∼77 °C. Based on the review of the literature given above, three questions pertaining to the conformations of avidin remain. (a) There is a disagreement on the presence/absence of R-helical secondary structure for avidin in solutions; (b) As a result, the interpretation of changes in secondary structure during avidin binding to biotin remains ambiguous; (c) Similarly, the interpretation of changes in secondary structure during thermal denaturation of avidin and avidin-biotin complex remains ambiguous. The experimental measurements of vibrational absorption and vibrational circular dichroism (VCD) along with ab initio calculations can provide a powerful approach for determining configuration and conformational behavior of chiral molecules.8 Both VCD9a-f and vibrational Raman optical activity (VROA)9g are useful for studying biomolecules. Recently, VCD has been used to investigate the structure of peptides, proteins, nucleic acids, and carbohydrates.9a-f The complicated nature of vibrational modes and overlapping vibrational bands generally pose problems in the interpretation of vibrational spectra. The amide I′ region represents an isolated region which provides useful information on the secondary structure of biological molecules. VCD can sense several different transitions involving different localized vibrations of the molecule. However, its use in the
10.1021/jp0104177 CCC: $20.00 © 2001 American Chemical Society Published on Web 07/12/2001
7858 J. Phys. Chem. B, Vol. 105, No. 32, 2001 elucidation of secondary structure of proteins is still limited to qualitative interpretations because of the difficulty in the quantum mechanical calculations on large molecules. Nevertheless, the availability of structurally well-defined synthetic peptides permits the assignment of specific VCD band patterns to unique conformational features. Recent VCD studies lead to the identification of characteristic VCD sign patterns for various secondary structures including R-helix, β-sheet, 310 helix, β-turns, and double helical structures in peptides/proteins.9a-f As mentioned earlier, the presence of R-helical secondary structure for avidin in liquid solutions remains controversial at the present time. VCD might be able to resolve this controversy because the presence of R-helical structure should reveal a unique characteristic VCD sign pattern. Vibrational circular dichroism has not been used before to study the secondary structure of avidin. The temperature effect on the secondary structure of this protein has not been investigated using VCD technique. In the present work, vibrational absorption and vibrational circular dichroism spectra have been measured for avidin and avidin-biotin complex. The effect of temperature on the secondary structure of avidin and its complex with biotin are elucidated by the combination of variable-temperature vibrational absorption and VCD spectroscopy. Two-dimensional (2D) correlation spectroscopic analysis,10 which is used to study the perturbations on the structure of biomolecules, is employed to generate correlation spectra (synchronous and asynchronous) based on a set of dynamic VCD spectra from temperatureinduced perturbations in the amide I′ region (CdO stretch vibration). Procedures Egg-white avidin and d-biotin were purchased from Sigma Chemical Co. (St. Louis, MO). D2O was from Cambridge Isotope Laboratories, Inc. The phosphate buffer (H2O solution, pH ) 7.2) was purchased from Aldrich. This buffer was mixed with D2O in 1:9 ratio, for exchanging hydrogen atoms with deuterium, and was used for preparing all solutions. Biotin solutions with concentrations of 0.15% (w/v) in the abovementioned buffer solution were used. For variable-temperature studies, avidin was dissolved in buffer solutions with (0.15% w/v) or without d-biotin to give a protein concentration of ∼10% (w/v). An aliquot of solution was placed between two CaF2 windows separated by a 50 µm Teflon spacer. The sample was then placed in a temperaturecontrolled IR cell. The infrared and VCD spectra were recorded in the 2000-900 cm-1 region on a commercial Fourier transform VCD spectrometer, Chiralir (Bomem-BioTools, Canada). All absorption spectra were collected by adding 512 scans at resolution of 8 cm-1, while the VCD spectra were recorded with 1 h data collection time at 8 cm-1 resolution in the temperature increase process. The temperature of the cell was controlled with an Omega temperature controller. The cell temperature was increased in 10 °C increments and allowed to equilibrate for 15 min prior to the acquisition of each spectrum. During the cooling cycle (20 °C increments for avidin and 10 °C increments for avidin-biotin complex), the VCD spectra were collected with 3 h data collection time at 8 cm-1 resolution. Curve fitting was undertaken with BGRAMS software using Lorentzian band shape. Fourier-deconvolved spectra were also obtained with BGRAMS software (gamma factor is 17.5, smoothing factor is 20%11,12). KG2D software written by Dr. Y. Wang and VCD spectra as a function of temperature were used to perform 2D-VCD correlation spectroscopy analysis.
Wang and Polavarapu
Figure 1. Stacked raw absorption spectra (35 °C) of (a) avidin (10% w/v), (b) avidin-biotin complex (10% w/v), and (c) difference absorption spectrum obtained by subtracting the avidin spectrum from the spectrum of the avidin-biotin complex.
Figure 2. Stacked VCD spectra (35 °C) of (a) avidin (10% w/v), and (b) avidin-biotin complex (10% w/v), and (c) difference VCD spectrum obtained by subtracting the avidin spectrum from the spectrum of the avidin-biotin complex.
Results and Discussion The absorption and VCD spectra of avidin are compared to those of avidin-biotin complex in Figures 1 and 2, respectively. Since the amount of biotin used is very small, its contributions to absorption and VCD are negligibly small. The differences between the spectra of avidin-biotin complex and avidin, as seen in both difference absorption (Figure 1c) and difference VCD (Figure 2c), are also small. Several absorption bands overlap in the amide I′ region, and as a consequence they cannot be easily discerned from raw absorption spectra. Curve-fitting or deconvolution techniques help resolve such overlapping bands. Curve fitting of absorption spectra (Figure 3) was undertaken after subtracting the buffer spectrum from the absorption spectra of sample. It is to be noted that the absorption spectra were recorded, along with the VCD spectra, at 8 cm-1 resolution so the band positions and the number of bands identified at this resolution can be slightly different from those identified from higher resolution absorption spectra in the literature. From the curve-fitting results (Figure 3a), four absorption bands were identified at 1693, 1674, 1653, and 1628 cm-1 for avidin. These band positions are in agreement with those reported by Swamy et al. (see Table 1). The band at 1693 cm-1 was assigned (see Table 1) to either antiparallel β-sheets or β-turns; the band at 1674 cm-1 was assigned to either turns, bends, or β-sheets; the band at 1653 cm-1 was assigned to either disordered structure or R-helices, and the band at 1628 cm-1 was assigned to antiparallel β-sheets or extended β-sheets. The VCD spectrum of avidin (Figure 2a) shows a weak doublet with positive band at 1693 cm-1 and negative band at 1674 cm-1
Avidin and Avidin-Biotin Complex
J. Phys. Chem. B, Vol. 105, No. 32, 2001 7859
Figure 3. Curve-fitting analysis of the amide I′ absorption spectra (the absorption spectrum of D2O buffer has been subtracted) of (a) avidin (35 °C); (b) avidin-biotin complex (35 °C), and (c) avidin (95 °C); squares (0) denote the curve-fitting results.
TABLE 1: Comparison of the VCD Observations with Literature Results literature 1f IR
assignment
literature 2g IR
assignment
literature 3h
present resultsa
Raman
assignment
IRb,d 1693 1682* 1677 1664 1650
1693 1682* 1674
1693 1682* 1674
1653
1645
1628
1628
1621
1619*
1620*
1612*
1694 1684 1674 1662 1650
sheet or turn sheet or turn turns & bends turns & bends R-helix
1694
β-turn
1696
β-sheet
1679
β-sheet
1656
disordered
1629
β-sheet or extended β-sheet
1630
β-sheet
1676 1665 1653 1645 1634
β-sheet random R-helix R-helix β-sheet
IRc,d
VCDd
IRe
assignment
1692
β-turn aggregated β-strand β-sheet turns & bends turns & bends
1679 1667 1651 1633 1618
β-sheet β-sheet aggregated β-strand
a
Asterisks(*) denote the bands observed at higher temperature. b From Fourier-deconvoluted spectra (gamma factor is 17.5 and smoothing factor is 20%); the bands at 1664 and 1650 cm-1 overlap and give one band at 1657 cm-1 if gamma factor 20 and smoothing factor 35% are used. c From curve-fitting results. d For avidin. e For avidin-biotin complex, and from curve-fitting results. f Ref 7. g Ref 3c. h Ref 3e.
and a strong doublet with positive band at 1645 cm-1 and negative band at 1621 cm-1. These VCD features are similar to those seen for chymotrypsin,9f which is known to have predominantly β-sheet structure. However, proteins with lefthanded R-helical structures, and random coil structures with a local left-handed twist, also give a negative VCD couplet9f (a bisignate couplet with positive VCD on the higher frequency side and negative VCD on the lower frequency side) in the 1650-1620 cm-1 region. Then it is necessary to distinguish between β-sheet, left-handed R-helical and random coil struc-
tures for avidin. For this purpose, we use the following reasoning. If both positive VCD at 1645 cm-1 and negative VCD at 1621 cm-1 in avidin originate from the same secondary structure (R-helix or random coil), then they are expected to change synchronously when that structure is perturbed, for example by increasing temperature. The absorption and VCD spectra of avidin in the amide I′ region, as a function of temperature between 35 and 95 °C, are shown in Figure 4a and 4b. The deconvoluted absorption spectrum (Figure 5a) shows five bands at 1693, 1677, 1664, 1650, and 1628 cm-1 at 35 °C.
7860 J. Phys. Chem. B, Vol. 105, No. 32, 2001
Wang and Polavarapu
Figure 4. Overlaid (a) raw absorption spectra and (b) VCD spectra of avidin in the 1800-1550 cm-1 region from 35 to 95 °C (increasing temperature in 10 °C intervals).
The bands at 1664 and 1650 cm-1 overlap each other and appear as one band at 1657 cm-1 if larger gamma factor and smoothing factor are used. The changes in peak intensities for Fourier deconvolved bands at 1628 and 1619 cm-1 of avidin as a function of temperature are shown in Figure 5b, while the changes in peak intensities for different VCD bands of avidin as a function of temperature are shown in Figure 6. The magnitude of negative VCD at 1621 cm-1 decreases gradually with increasing temperature until about 65 °C, then changes the rate of variation between 65 °C and 85 °C and remains constant beyond 85 °C. This pattern of variation, similar to that obtained for Fourier deconvoluted absorption bands (Figure 5b), indicates a cooperative transition in the secondary structure at about 75 °C (from VCD) or 80 °C (from absorption). At this transition, the position of negative VCD band shifts from 1621 to 1612 cm-1. The behavior of positive VCD at 1645 cm-1, however, is different from that at 1621 cm-1 (see Figures 4b and 6a). The magnitude of positive VCD band at 1645 cm-1 remains nearly constant until about 75 °C and then vanishes beyond this temperature. Since the temperature-dependent variations of VCD at 1645 and 1621 cm-1 are entirely different, it is reasonable to conclude that these two bands do not originate from a left-handed R-helical structure or a random coil structure with local left-handed twist. Beyond 95 °C, VCD spectrum of avidin shows just two negative VCD bands, one at 1612 cm-1 and another at 1682 cm-1, and these bands remain unaffected while the temperature is lowered to 35 °C (see Figure 7a,b). These two widely separated negative bands are a characteristic feature of aggregated antiparallel β-strands.9f These observations indicate that as the temperature is increased, avidin undergoes an irreversible transition from β-sheet structure (characterized by negative VCD bands at 1621 and 1674 cm-1) to aggregated structure that contains antiparallel β-strands (characterized by negative VCD bands at 1612 and 1682 cm-1). The two positive VCD bands of avidin at 1645 and 1693 cm-1, which collapse
Figure 5. (a) Fourier deconvolved spectra of avidin in the 17001600 cm-1 region from 35 to 95 °C; (b) temperature dependence of the peak heights of the 1628 and 1619 cm-1 absorption bands in the Fourier deconvolved spectra of avidin (10% w/v).
beyond the transition temperature, are likely to originate from turns/bends in the native avidin structure. The addition of small amounts of biotin is known1,2 to result in a remarkable stabilization of avidin against thermal denaturation. This known fact is also supported by the present results. Curve-fitting results (Figure 3b and Table 1) indicate that the absorption bands at 1693 and 1653 cm-1 remained as in avidin. But in place of one absorption band at 1674 cm-1 in avidin, two absorption bands are seen for avidin-biotin at 1679 and 1667 cm-1. Also in place of one absorption band at 1628 cm-1 in avidin, two absorption bands at 1633 and 1618 cm-1 are seen for avidin-biotin. These changes were suggested7 to arise from the formation of a β-sheet structure which is more shielded from solvent. The VCD spectrum of avidin-biotin complex (Figure 2b) contains a strong couplet, just like avidin, with a positive band at 1645 cm-1 and a negative band at 1621 cm-1. The weak VCD couplet seen for avidin with a positive band at 1693 cm-1
Avidin and Avidin-Biotin Complex
J. Phys. Chem. B, Vol. 105, No. 32, 2001 7861
Figure 7. Overlaid (a) raw absorption spectra of avidin from 95 to 35 °C (cooling cycle at 20 °C intervals) and (b) VCD spectra of avidin from 95 to 35 °C (cooling cycle at 20 °C intervals), in the 1800-1550 cm-1 region.
Figure 6. Temperature dependence of the peak heights of the amide I′ bands in the VCD spectra of (a) avidin (10% w/v) and (b) avidinbiotin complex (10% w/v), asterisk (*) on band position denotes changes in cooling cycles.
and a negative band at 1674 cm-1 is also present in the VCD spectrum of avidin-biotin complex, although not as pronounced. The changes in absorption spectra and near equivalence of VCD spectra in avidin and avidin-biotin complex indicate that the presence of biotin is introducing more nonequivalent amide groups (as a result of binding biotin to avidin), although the overall structural arrangement is not drastically different. As the temperature is increased, the intensity of negative VCD at 1621 cm-1 in avidin-biotin complex decreases (see Figures 6b and 8), indicating some unfolding of β-sheet structure. However, unlike in avidin, neither a cooperative structural transition nor aggregation takes place in avidin-biotin complex as revealed by the noncollapsing VCD bands (at 1621 and 1645 cm-1) in the latter even at 95 °C. Nevertheless, some reversible unfolding does take place in avidin-biotin complex during the heating process, because the changes that take place during
Figure 8. Overlaid (a) raw absorption spectra and (b) VCD spectra of avidin-biotin complex in the 1800-1550 cm-1 region from 35 to 95 °C (increasing temperature in 10 °C intervals).
heating (see Figure 8) do revert during the cooling process (see Figure 9). These reversible changes can also be seen in the absorption spectra when they are deconvoluted, as reported in ref 7, although they are not clearly apparent in the raw absorption spectra.
7862 J. Phys. Chem. B, Vol. 105, No. 32, 2001
Figure 9. Overlaid (a) raw absorption spectra of avidin-biotin from 95 to 35 °C (cooling cycle at 10 °C interval) and (b) VCD spectra of avidin-biotin from 95 to 35 °C (cooling cycle at 10 °C interval), in the 1800-1550 cm-1 region.
H-D exchange can also contribute to the temperaturedependent intensity changes in the amide I and amide II regions. The amide II region is sensitive to the H-D exchange while the amide I is much less sensitive to it. This issue has been addressed before in the literature.7,13 However, there are some differences between the present study and those in the literature. First the buffer, prepared as H2O solution, is mixed with excess D2O to facilitate the exchange of hydrogen atoms in the buffer with deuterium. Second, the initial spectra were taken by maintaining the solution temperature at 35 °C (instead of 20 °C) and data collection time for VCD is longer than that for FTIR. Under these conditions, our spectra (Figures 10a and 10b) for avidin do not indicate the presence of any significant amide II bands (at about 1550 cm-1), but they are replaced by amide II′ bands (at about 1450 cm-1). This indicates that no significant amount of unexchanged protons is present in avidin even at 35 °C. In the case of avidin-biotin complex, however, weak amide II bands (at about 1550 cm-1) are present even at higher temperatures, indicating only a partial H-D exchange. Nevertheless, the temperature-dependent absorption or VCD intensity changes seen in amide I′ region for avidin and avidin-biotin complex are not considered to originate from H-D exchange for the following reasons. Temperature-dependent absorption intensity changes are seen in the amide II′ region (at ∼1450 cm-1) for avidin-biotin complex (Figures 10c and 10d), but similar intensity changes are seen for buffer itself (Figures 10e and 10f). When the buffer spectra are subtracted, we find that temperature-dependent amide II′ absorption intensity changes are not apparent in the cooling cycle, but small increases in the amide II′ absorption intensities are evident in the heating cycle. These amide II′ absorption intensity changes, however, appear to be independent of the temperature-dependent VCD intensity changes in the amide I′ region, because the latter are reversible (Figures 8 and 9). These observations suggest that H-D exchange is not a significant contributor to the observed
Wang and Polavarapu temperature-dependent VCD intensity changes in the amide I′ region of avidin-biotin complex. In the case of avidin, as stated earlier, H-D exchange appears to be complete, as inferred by the absence of any significant amide II bands, even at 35 °C. Thus the temperature-dependent VCD intensity changes seen in the amide I′ region for avidin are considered to be originating from the corresponding structural variations in the protein. Analysis of the effect of temperature on the amide I′ bands in the VCD spectra of avidin by 2D-VCD correlation spectroscopy is shown in Figure 11. Similar analysis on the temperaturedependent absorption spectra of avidin was reported in ref 7. In the synchronous 2D correlation map, the auto correlation peaks (peaks on the diagonal line) represent10 the extent of spectral intensity variation. Thus the autopeaks in the synchronous 2D-VCD correlation map (Figure 11a) at 1645 and 1621 cm-1 indicate that major intensity changes occur at these positions during the increase of temperature. Fewer number of lines at the 1645 autopeak is indicative of abrupt variation of of VCD intensity of this band with the change of temperature. Autopeaks are not seen at 1674 and 1693 cm-1, probably because these VCD bands are weak and their variations are accordingly small. The cross correlation peaks in a synchronous map represent10 simultaneous changes in spectral intensities of two different bands. In the case of VCD, unlike in absorption spectra, bands can be positive or negative. So it is necessary to identify the positive and negative changes in relation to the sign of VCD bands. For example, a decrease in the magnitude of negative VCD band intensity (a positive change) and an increase in the magnitude of positive VCD band intensity (a positive change) would be registered as a positive cross-peak in the correlation map. Similarly a decrease in the magnitude of positive VCD band intensity (a negative change) and an increase in the magnitude of negative VCD band intensity (a negative change) would be registered as positive cross-peak in the correlation map. Thus a negative cross correlation peak at 1621 vs 1645 cm-1 would indicate that as the magnitude of negative VCD intensity of the 1621 cm-1 band decreases (a positive change), the magnitude of positive VCD intensity of the 1645 cm-1 band decreases (a negative change). The same explanation applies to the cross correlation peak at 1621 vs 1693 cm-1, since the 1693 cm-1 band also has positive VCD band intensity. However since the VCD band at 1682 cm-1 is negative, as is the band at 1621 cm-1, a negative cross correlation peak at 1621 vs 1682 cm-1 indicates that these two band intensities change in opposite directions (i.e., as the magnitude of negative VCD intensity at 1621 cm-1 decreases, the magnitude of negative VCD band intensity at 1682 cm-1 increases). The positive cross correlation peak at 1621 vs 1674 cm-1 indicates that the magnitude of negative VCD band intensity at 1674 cm-1 decreases along with that of negative VCD band at 1621 cm-1. The negative cross correlation at 1645 vs 1674 cm-1 indicates that the magnitudes of both VCD bands decrease since the former is positive while the latter is negative. In the asynchronous 2D-correlation map (Figure 11b), only cross correlation peaks appear (i.e., no diagonal peaks) and it is antisymmetric (i.e., the peaks above the diagonal line are opposite to those below the diagonal line). The cross correlation peaks here indicate10 the time sequence (representing temperature, pressure, or concentration-based perturbation) of changes in intensities of bands at ν1 and ν2. A positive cross correlation peak at ν1 vs ν2 above the diagonal line indicates that the intensity changes at ν1 occur at an earlier time than those at ν2. A negative cross correlation peak would indicate the reverse order. In asynchronous 2D-VCD correlation map (Figure 11b),
Avidin and Avidin-Biotin Complex
J. Phys. Chem. B, Vol. 105, No. 32, 2001 7863
Figure 10. Overlaid raw absorption spectra in the amide I′ and amide II′ regions of (a) avidin from 35 to 95 °C (increasing temperature in 10 °C intervals); (b) avidin from 95 to 35 °C (decreasing temperature in 20 °C intervals); (c) avidin-biotin complex from 35 to 95 °C (increasing temperature in 10 °C intervals); (d) avidin-biotin complex from 95 to 35 °C (decreasing temperature in 10 °C intervals); (e) phosphate buffer from 35 to 95 °C (increasing temperature in 10 °C intervals); and (f) phosphate buffer from 95 to 35 °C (decreasing temperature in 10 °C intervals)
a negative cross correlation peak above the diagonal line at 1612 vs 1621 cm-1 suggests the VCD intensity changes at 1612 cm-1 occur at a higher temperature than those at 1621 cm-1. The positive cross correlation peak at 1621 vs.1645 cm-1 above the diagonal line indicates the VCD intensity changes at 1621 cm-1 occur at lower temperature than those at 1645 cm-1. The same explanation applies to positive cross correlation peak at 1621 vs 1682 cm-1 and at 1645 vs 1693 cm-1 (above the diagonal line). Thus, as the temperature is increased, the β-sheet structure
of avidin is unfolded first, then turns and bends are disrupted, leading to the formation of aggregated antiparallel β-strand structure. Summary The results of curve-fitting and deconvoluted results of vibrational absorption spectra, variable-temperature absorption and VCD spectra, and 2D-VCD correlation analysis indicate
7864 J. Phys. Chem. B, Vol. 105, No. 32, 2001
Wang and Polavarapu ible unfolding as the temperature is increased, but does not undergo cooperative structural transition. 2D-VCD correlation spectroscopy is used to provide information on the sequence of events involved in protein unfolding, which reveals the disruption of β-sheet structure of avidin first and then the formation of intermolecular antiparallel β-strand, leading to aggregation of the protein. Acknowledgment. We thank Professor Yukihiro Ozaki and Dr. Yan Wang for providing us with Array Basic program KG2D used to calculate the 2D correlation spectra. Grants from NSF (CHE9707773) and Vanderbilt University are gratefully acknowledged. References and Notes
Figure 11. 2D-VCD (a) synchronous and (b) asynchronous spectra constructed from the VCD spectral data in Figure 4b. Negative cross correlation peaks are shown as dashed lines and positive crosscorrelation peaks are shown as full lines.
that (a) Avidin does not contain any significant amount of R-helical structure; its structure is predominantly β-sheet with some turns and bends; (b) At about 75-80 °C, avidin undergoes irreversible cooperative structural transition to aggregated antiparallel β-strands; (c) The structure of avidin-biotin complex is not significantly different from that of avidin, but binding of biotin increases the number of nonequivalent amide groups; (d) Avidin-biotin complex does undergo some revers-
(1) (a) Savage, M. D.; Matton, G.; Desai, S.; Mielander, G. W.; Morgensen, S.; Conklin, E. J. AVidin-Biotin Chemistry: A Handbook; Pierce: Rockford, IL, 1992. (b) Wilchek, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1-32. (c) Wilchek, M.; Bayer, E. A. In Protein Recognition of Immobilized Ligands; Hutchins, T. W., Ed.; Alan R. Liss, Inc: New York, 1989; pp 83-90. (d) Henrikson, K. P.; Allen, S. H. G.; Maloy, W. L. Anal. Biochem. 1979, 94, 336. (2) (a) Green, N. M. Methods Enzymol. 1990, 184, 51. (b) Green, N. M. Biochem. J. 1963, 89, 585. (3) (a) Honzatko, R. B.; Williams, P. W. Biochemistry 1982, 21, 6201. (b) Barbucci, R.; Magnani, A.; Roncolini, C.; Silvestri, S. Biopolymer 1991, 21, 827. (c) Swamy, M. J.; Heimburg, T.; Marsh, D. Biophys. J. 1996, 71, 840. (d) Fagnano, C.; Fini, G.; Torreggiani, A. J. Raman Spectrosc. 1995, 26, 991. (e) Torreggiani, A.; Fini, G. J. Raman Spectrosc. 1998, 29, 229. (4) (a) Livnah, O.; Bayer, E. A.; Wilchek, M.; Sussman, J. L. Proc. Natl. Acad. Sci. 1993, USA90, 5076. (b) Pugliese, L.; Coda, A.; Malcovati, M.; Bolognes, M. J. Mol. Biol. 1993, 231, 698. (c) Green, N. M.; Melamed, M. D. Biochem. J. 1966, 100, 614. (5) Donovan, J. W.; Ross, K. D. Biochemistry 1973, 12, 512. (6) Green, N. M. Biochem. J. 1963, 89, 609. (7) Ismoyo, F.; Wang, Y.; Ismail, A. A. Appl. Spectrosc. 2000, 54, 939. (8) (a) Aamouche, A.; Devlin, F. J.; Stephens, P. J. J. Am. Chem. Soc. 2000, 122, 2346. (b) Ashvar, C. S.; Devlin, F. J.; Stephens, P. J. J. Am. Chem. Soc. 1999, 121, 2836. (c) Polavarapu, P. L.; Zhao, C.; Ramig, K. Tetrahedron: Asymmetry 1999, 10, 1099. (d) Polavarapu, P. L.; Zhao, C.; Cholli, A. L.; Vernice, G. J. Phys. Chem. 1999, 103, 6127. (e) Wang, Feng; Polavarapu, P. L.; Drabowicz, Jozef; Mikoajczyk, M. J. Org. Chem. 2000, 65, 7561. (9) (a) Zhao, C.; Polavarapu, P. L.; Das, C.; Balaram, P. J. Am.. Chem. Soc. 2000, 122, 8228. (b) Zhao, C.; Polavarapu, P. L. J. Am. Chem. Soc. 1999, 121, 11259. (c) Xie, P.; Zhou, Q.; Diem, M. J. Am. Chem. Soc. 1995, 117, 9502. (d) Tam, C. N.; Bour, P.; Keiderling, T. A. J. Am. Chem. Soc. 1996, 118, 10285. (e) Yu, G.-S.; Che, D.; Freedman, T. B.; Nafie, L. A. Biospectroscopy 1995, 1, 113. (f) Bour, P.; Keiderling, T. A. J. Am. Chem. Soc. 1993, 115, 9602. (g) Hecht, L.; Barron, L. D.; Blanch, E. W.; Bell, A. F.; Day, L. A. J. Raman. Spectrosc. 1999, 30, 815. (10) Noda, I. Appl. Spectrosc. 1993, 47, 1329. (11) Surewicz, W. K.; Mantsch, H. H.; Chapman, D. Biochemistry 1993, 32, 389. (12) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 469. (13) Meskers, S.; Ruysschaert, J. M.; Goormaghtigh, E. J. Am. Chem. Soc. 1999, 121, 5115.
