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Two-dimensional Fourier-transform infrared correlation spectroscopy study of the high-pressure tuning of proteins
Vibrational Spectroscopy 22 Ž2000. 119–125 www.elsevier.comrlocatervibspec
Two-dimensional Fourier-transform infrared correlation spectroscopy study of the high-pressure tuning of proteins L. Smeller a
a,)
, P. Rubens b, J. Frank c , J. Fidy a , K. Heremans
b
Institute of Biophysics and Radiation Biology, Semmelweis UniÕersity of Medicine, Budapest, Puskin u. 9 r P.O. Box 263, H-1444 Hungary b Department of Chemistry, Katholieke UniÕersiteit LeuÕen, B-3001 LeuÕen, Belgium c KluyÕer Laboratory of Biotechnology, Delft UniÕersity of Technology, 2628 BC Delft, Netherlands Received 5 May 1999; received in revised form 18 June 1999; accepted 25 June 1999
Abstract 2D FTIR gives new information about the pressure effect on the structure and dynamics of macromolecular systems. Application of this analysis to proteins can unravel the relation of conformational changes and HrD exchange processes. For lipoxygenase, a pressure of 6.5 kbar induces irreversible conformational changes resulting in an increased exposure of interior parts of the protein to the solvent. At the transition pressure the spectral changes indicate a correlation between conformational changes and HrD exchange. Below and above this pressure, the effects of HrD exchange on the spectral changes are predominant. q 2000 Elsevier Science B.V. All rights reserved. Keywords: HrD exchange; Proteins; FTIR correlation spectroscopy
1. Introduction Fourier-transform infrared spectroscopy ŽFTIR. has recently been used for the in situ secondary structure determination of pressurized proteins w1–3x. The development of the two-dimensional Ž2D. correlation spectroscopy w4x opened new possibilities in this field. This new technique is a powerful tool for studying spectral changes induced by external perturbation. Originally the method was developed for sinusoidally varying external perturbations like mechanical stress in polymers. The 2D correlation spec-
) Corresponding author. Fax: q36-1-266-6656; e-mail: [email protected]
troscopy was later generalized by its developer Noda w5x for nonsinusoidal and even for nonperiodic perturbations. 2D IR analysis was used to study the melting of nylon 12 w6x, the solvent-induced perturbation of protein conformation w7x, temperature-induced protein unfolding w8x, but pressure-induced unfolding of proteins is quite new in this line w9x. One can extract additional information, from a 2D analysis of vibrational spectra, because it reflects the response of the system to the external perturbation. Correlation between changes at different spectral positions can be obtained. If overlapping subcomponents of a complex band reply to the external perturbation in different ways, resolution enhancement can also be achieved. These features allow a detailed investigation of the effect of the perturbing parame-
0924-2031r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 4 - 2 0 3 1 Ž 9 9 . 0 0 0 3 7 - 5
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L. Smeller et al.r Vibrational Spectroscopy 22 (2000) 119–125
ter on the inter- as well as on the intra-molecular interactions. We apply the method to separate overlapping spectral effects due to hydrogen–deuterium ŽHrD. exchange and to changes in the protein backbone conformation. Proteins are stable in their native structure only in very limited temperature and pressure range w10x. According to a general thermodynamic theory, this range is an elliptic area on the p–T space w11,12x. This means that in addition to the well-known heat denaturation, proteins can be unfolded by subjecting them to pressure or cooling. The difference between the effects of high pressure, high temperature and low temperature on the protein structure has been investigated by NMR w13x and UV difference spectroscopy w14x. Nevertheless, there remains the need to follow the changes in secondary structure of proteins under these conditions. The only approach that is available at present is vibrational spectroscopy w10x. In this paper, we used high pressure as perturbing tool for the 2D FTIR spectroscopy in combination with the high pressure diamond anvil cell. The method is demonstrated here using soybean lipoxygenase. Lipoxygenase is a large single-chain protein composed of 839 amino acids. Its structure is composed of two domains: the N-terminal b-barrel is associated with one helix and the major domain is composed of 22 helices and 8 b-strands w15x. The crystal structure of soybean lipoxygenase L-1 is available at 1.4 A resolution w16x. The pressure and temperature inactivation of this enzyme is also of considerable practical interest in view of its role in the quality preservation of food products w17,18x.
2. Experimental Preparation of soybean lipoxygenase is described elsewhere w19x. The protein was dissolved in 10 mM Tris at pD 7.8 in the solvent D 2 O using a protein concentration of 30 mgrml. The solution was left overnight at room temperature and ambient pressure to allow the HrD exchange of the easily accessible and fast exchanging protons. In order to study the kinetics of the slow exchanging protons the pressure cell was thermostated Žafter filling it and applying a
very low pressure to avoid leakage.. Once the chosen temperature was obtained, the sample was pressurized. This was usually done within 4 min. Kinetic measurements were started when the desired pressure was reached. The spectra used for the 2D analysis were taken during a period of 2 h. Infrared spectra were recorded with a Bruker IFS66 FTIR spectrometer equipped with a broadband MCT detector. A total of 250 interferograms were coadded after registration at a resolution of 2 cmy1 . High pressure was generated in a diamond anvil cell ŽDiacell Products, Leicester, UK. and the temperature was measured by a thermocouple. The gasket between the diamonds had an original thickness of 75 mm, which resulted in a sample thickness of approximately 50 mm. The pressure was determined by an internal calibrant, BaSO4 , using the shift of the 983 cmy1 peak w20x. The 2D correlation spectra were calculated by software developed in our laboratory, using the equations of Noda w5x. The reference spectrum is the average for each of the three pressure regions discussed below. For the lower and higher pressure regions a total of eight spectra were used to calculate the correlation spectra. Sixteen spectra were used for the transition region. The spectra were taken in almost equidistant pressure steps of 0.3 " 0.1 kbar. No spectral manipulations Žbackground substraction or resolution enhancement. were performed prior to the 2D analysis. For the representation of the 2D correlation spectra we use the following convention. In a synchronous spectrum, intensity changes observed at two spectral coordinates in opposite direction are indicated by shaded negative crosspeaks. Positive crosspeaks without shading indicate changes in the same direction. In the asynchronous spectrum, the negative peaks are shaded. The interpretation of the sign of the spectral peaks in the asynchronous spectrum is more complex. If a spectral peak at n 1 , n 2 wavenumbers has a positive sign, the spectral changes at n 1 precede the intensity changes at the wavenumber n 2 . This rule is however reversed, if the synchronous plot at the corresponding n 1 , n 2 position has a negative value. The notation of n 1 and n 2 follows the equations of Noda w4,5x. In the standard 2D plots n 1 is on the horizontal axis and n 2 on the vertical one.
L. Smeller et al.r Vibrational Spectroscopy 22 (2000) 119–125
3. Results and discussion Fig. 1 shows the amide IrIX , II and IIX regions of the FTIR spectra in the pressure range of 1–11 kbar. I and II refer to the protonated while IX and IIX refer to the deuterated amide group. The amide I vibration is mainly a C5O stretching, with some small contribution from the N–H bending. It appears between 1600 and 1700 cmy1 , depending on the actual conformation of the protein. In a folded protein the oxygen atom is hydrogen-bonded to the other parts of the polypeptide chain. Therefore, the amide I vibration is sensitive to the secondary structure. Subcomponents of this band can be assigned to different secondary structures w21x, while similar assignments for the amide II Žand IIX . vibrations are less clear-cut. The amide II Žand IIX . modes are mixed vibration modes involving N–H ŽN–D. bending and C–N stretching. Since this vibration strongly involves the hydrogen Žor deuterium. atom the band shifts ca. 100 wavenumbers down upon deuteration w22x. Its position in the protonated protein is at about 1550 cmy1 . The exact position depends only slightly on the specific secondary structure. If the wavenumber maximum of the amide I band is plotted vs. pressure, a sigmoid curve is obtained
X
X
121
with the transition midpoint at 6.5 kbar which can be treated as the denaturation pressure w19x. The wavenumber shift alone, however, is very misleading in this case, because it is not a true shift, but an apparent one, resulting from the relative intensity changes of the subcomponents of the broad amide I band. Analysis of the band with curve fitting is problematic as part of the shifts is due to HrD exchange. Since the spectra do not show very marked changes, we turn to 2D analysis of the data, to gain more detailed information about the conformational change, the HrD exchange and the possible coupling between these processes. The pressure range was divided into three parts for the 2D correlation analysis: low pressure range Ž2–4 kbar., the transition region Ž4.7–9.0 kbar., and the upper pressure region Ž8.5–10.6 kbar.. Fig. 2 shows the synchronous and asynchronous spectra of lipoxygenase for the low pressure range. The synchronous spectrum in the low pressure region showed intense auto and crosspeaks in the amide IIX region Ž1472, 1391 cmy1 . while very minor changes can be seen in the amide I band. The peaks of amide IIX region are very sensitive to HrD exchange. However, since there are no negative
Fig. 1. Amide IrI , II and II regions of the FTIR spectra of soybean lipoxygenase in the pressure range of 1–11 kbar. Pressure increases from bottom to top in equal pressurertime steps.
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L. Smeller et al.r Vibrational Spectroscopy 22 (2000) 119–125
Fig. 2. Synchronous Ža. and asynchronous Žb. spectra of lipoxygenase for the low pressure range Ž2.3–4.4 kbar.. The negative peaks are shaded. The reference spectrum can be seen on the side of the 2D plot.
crosspeaks for the amide II and amide IIX bands, the peaks at 1472 and 1391 cmy1 can also reflect the slight elastic deformation of the protein. Unfortunately, the assignment of the components of the amide IIX band to the different secondary structures is not as simple as for the amide I band. Therefore, we cannot assign these changes to any part of structural component of the protein. The small autopeak at 1620 cmy1 might indicate the increase of the intermolecular interactions, while the negative crosspeak at 1472 and 1655 cmy1 indicates some loss in the helical structure. It is of interest to note that the active site is located in the domain that contains all the helices w16x. The asynchronous plot indicates that the spectral changes at 1620 cmy1 precede the amide IIX changes. The 2D spectra obtained in the transition region Ž4.5–9 kbar. can be seen on Fig. 3. The synchronous spectrum is much better populated by correlation peaks than the former plot was. Conformational changes and exchange effects are both present in these spectra. The following spectral bands show remarkable contribution to the 2D spectra: 1686, 1652, 1619, 1541, 1475, 1424 and 1365 cmy1 . The
spectral changes in the amide I region reflect the unfolding of the protein. The decrease of the folded structure can be seen from the peak at 1655, which is anticorrelated to the increasing amide IIX band and positively correlated to the diminishing amide II band. The anticorrelation between the 1655 and 1620 bands might indicate further increasing intermolecular interactions. The amide II and IIX bands anticorrelate as one would expect. The analysis of the asynchronous spectrum results in the following sequence of intensity changes of the main peaks: 1620 cmy1 Žamide IrIX . 1473 cmy1 Žamide IIX . 1650 cmy1 Žamide IrIX . 1540 cmy1 Žamide II.. The other peaks change in a manner uncorrelated with the above ones. This sequence shows that the conformational changes of the protein are correlated with the HrD exchange. Loosening of the original conformation enhances the exchange, while the exchanged protein undergoes further unfolding and loss of secondary structure. The pressure region where the protein is already unfolded gives the 2D spectra which can be seen in Fig. 4. Above 8.5 kbar the protein lost its enzymatic function completely, but fragments of the secondary
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L. Smeller et al.r Vibrational Spectroscopy 22 (2000) 119–125
123
Fig. 3. Synchronous Ža. and asynchronous Žb. spectra of lipoxygenase for the transition regime Ž4.7–9.0 kbar.. The negative peaks are shaded. The reference spectrum can be seen on the side of the 2D plot.
structure are still present. This can be seen from the autopeak in the synchronous spectrum at 1640 cmy1
which reflects the further increase of the unordered chain. The remaining ordered structure of the protein
Fig. 4. Synchronous Ža. and asynchronous Žb. spectra of lipoxygenase in the high pressure region Ž8.5–10.6 kbar.. The negative peaks are shaded. The reference spectrum can be seen on the side of the 2D plot.
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L. Smeller et al.r Vibrational Spectroscopy 22 (2000) 119–125
becomes smaller by further increasing the pressure in this high pressure range. Consequently the less accessible protons will also exchange. This can be seen in the amide II and IIX region by observing the auto and crosspeaks at 1380, 1460, 1525 and 1550 cmy1 . Since there is again a band in the amide II Ž1525 cmy1 . which is not anticorrelated with the amide IIX ones, we assign it to the conformational changes in the folded fragments, instead of the HrD exchange. In the asynchronous spectrum we note that the amide II and IIX changes Žat 1380, 1460, 1525 cmy1 . follow the weak spectral changes at 1640 cmy1 associated to the unordered structure. From the previous analysis, it can be noted that the position of the correlation peaks for the same band differs slightly for the different pressure regions. This may be attributed to pressure-induced frequency shifts, pressure-induced distortions of the secondary structural elements of the protein or a combination of both effects w23x. In order to clarify further the connection between the exchange and conformational changes, kinetic experiments were performed slightly below and above the transition pressure Ž6.5 kbar.. Since the fast accessible protons were already exchanged, Žsee Section 2 for the details of the kinetic measurements., the exchange observed after the pressure jump can be attributed to the protons that are exposed to the solvent due to the pressure induced conformational alterations in the protein structure. Fig. 5 shows the synchronous spectra obtained at Ža. 5.2 and Žb. 7.3 kbar. After a pressure jump to 5.2 kbar, the main peak can be seen in the amide I region. This means that some conformational changes started already at this pressure. Small crosspeaks at 1650, 1475 cmy1 and at 1650, 1545 cmy1 indicate the small exchange induced by the loosening of the conformation. Since the amide IIrIIX bands are very sensitive to the HrD exchange, the small intensity of the peaks indicates that the number of protons that are exposed to the D 2 O solvent due to the jump to 5.2 kbar is very small. This means that the conformation of the protein did not change considerably, and only a few hydrogen atoms became exposed to the solvent. Fig. 5b shows the synchronous spectrum of the kinetic experiment at 7.2 kbar. The fact that the amide II and IIX auto- and crosspeaks dominate the spectrum, shows that the number of protons accessi-
Fig. 5. Synchronous spectra of kinetic experiments on lipoxygenase at high pressure Ža.: at 5.2 kbar, Žb. at 7.3 kbar. The negative peaks are shaded. The reference spectrum can be seen on the side of the 2D plot.
ble to the solvent is increased by pressure-induced unfolding of the protein. The absolute intensities of the amide II and IIX autopeaks are five times higher on this picture compared to the 5.2 kbar one, where the autopeaks are not visible due to their very low intensity. Since the kinetic experiment was started
L. Smeller et al.r Vibrational Spectroscopy 22 (2000) 119–125
after reaching the desired pressure, the relative small intensity of the amide I peak suggests, that the unfolding of the protein is a fast process, the equilibrium conformation was reached in 3–4 min. This was the time required for increasing the pressure to the desired value. The conformational changes were completed before the start of the kinetic experiment, that is why the 2D spectrum mainly reflects the slower HrD exchange process induced by unfolding. It is of interest to compare the differences between a small protein, such as bovine pancreatic trypsin inhibitor ŽBPTI, 8 kDa. and lipoxygenase, which is a large protein Ž102 kDa.. For BPTI we observe an increased rate of HrD exchange below 5 kbar w2x. Above that pressure only a conformational change takes place. For BPTI it seems that the low pressure regime acts preferentially on the conformational dynamics thus promoting the HrD exchange. In the case of lipoxygenase, pressure induces irreversible conformational changes resulting in an increased exposure of interior parts of the protein to the solvent. Summarizing, we can conclude that the application of pressure as a perturbing parameter to 2D FTIR gives new information about the pressure effect on the structure and dynamics of macromolecular systems. Application of this analysis to biological systems like proteins can unravel complex phenomena like the relation of conformational changes and HrD exchange processes. These effects are difficult to separate in the conventional one-dimensional spectra. Overlapping bands of amide I region can be separated under these conditions when they respond to the perturbation in different ways.
Acknowledgements This research was supported in part by an EU project ŽFAIR CT96-1175., FWO, Flanders, the
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Hungarian Academy of Sciences, Budapest, the Ministry of Culture and Education, Hungary ŽMKM FKFP 1191r97. and a COST D10 action. References w1x N. Takeda, M. Kato, Y. Taniguchi, Biochemistry 34 Ž1995. 5980. w2x K. Goosens, L. Smeller, J. Frank, K. Heremans, Eur. J. Biochem. 236 Ž1996. 254. w3x G. Panick, R. Malessa, R. Winter, G. Rapp, K.J. Frye, C.A. Royer, J. Mol. Biol. 275 Ž1998. 389. w4x I. Noda, Appl. Spectrosc. 44 Ž1990. 550. w5x I. Noda, Appl. Spectrosc. 47 Ž1993. 1329. w6x Y. Ozaki, Y. Liu, I. Noda, Macromolecules 30 Ž1997. 2391. w7x N.L. Sefara, N.P. Magtoto, H.H. Richardson, Appl. Spectrosc. 51 Ž1997. 536. w8x C.P. Schultz, H. Fabian, H.H. Mantsch, Biospectroscopy 4 Ž1998. S19. w9x L. Smeller, K. Heremans, Vib. Spectrosc. 19 Ž1999. 377. w10x K. Heremans, L. Smeller, Biochim. Biophys. Acta 1368 Ž1998. 353. w11x K. Suzuki, Rev. Phys. Chem. Jpn. 29 Ž1960. 91. w12x S.A. Hawley, Biochemistry 10 Ž1971. 2436. w13x J. Zhang, X. Peng, A. Jonas, J. Jonas, Biochemistry 34 Ž1995. 8361. w14x E. Mombelli, M. Afshar, P. Fusi, M. Mariani, P. Tortora, J.P. Connelly, R. Lange, Biochemistry 36 Ž1997. 8733. w15x B.J. Gaffney, Annu. Rev. Biophys. Biomol. Struct. 25 Ž1996. 431. w16x W. Minor, J. Steczko, B. Stec, Z. Otwinski, J.T. Bolin, R. Walter, B. Axelrod, Biochemistry 35 Ž1996. 10687. w17x K. Heremans, J. Van Camp, A. Huyghebaert, in: A. Paraf, S. Damodaran ŽEds.., Food Proteins and Their Applications, Marcel Dekker, 1996, p. 473. w18x M. Hendrickx, L. Ludikhuyze, I. Van den Broeck, C. Weemaes, Trends Food Sci. Technol. 8 Ž1998. 197. w19x O. Heinisch, E. Kowalski, K. Goossens, J. Frank, K. Heremans, H. Ludwig, B. Tauscher, Z. Lebensm.-Unters. Forsch. 201 Ž1995. 562. w20x P.T.T. Wong, D.J. Moffat, Appl. Spectrosc. 43 Ž1989. 1279. w21x D.M. Byler, H. Susi, Biopolymers 25 Ž1986. 469. w22x P.T.T. Wong, Can. J. Chem. 69 Ž1991. 1699. w23x K. Heremans, K. Goossens, L. Smeller, in: J.L. Markley, D.B. Northrop, C.A. Royer ŽEds.., High Pressure Effects in Molecular Biophysics, Oxford Univ. Press, NY, 1996, p. 44.
Two-dimensional Fourier-transform infrared correlation spectroscopy study of the high-pressure tuning of proteins L. Smeller a
a,)
, P. Rubens b, J. Frank c , J. Fidy a , K. Heremans
b
Institute of Biophysics and Radiation Biology, Semmelweis UniÕersity of Medicine, Budapest, Puskin u. 9 r P.O. Box 263, H-1444 Hungary b Department of Chemistry, Katholieke UniÕersiteit LeuÕen, B-3001 LeuÕen, Belgium c KluyÕer Laboratory of Biotechnology, Delft UniÕersity of Technology, 2628 BC Delft, Netherlands Received 5 May 1999; received in revised form 18 June 1999; accepted 25 June 1999
Abstract 2D FTIR gives new information about the pressure effect on the structure and dynamics of macromolecular systems. Application of this analysis to proteins can unravel the relation of conformational changes and HrD exchange processes. For lipoxygenase, a pressure of 6.5 kbar induces irreversible conformational changes resulting in an increased exposure of interior parts of the protein to the solvent. At the transition pressure the spectral changes indicate a correlation between conformational changes and HrD exchange. Below and above this pressure, the effects of HrD exchange on the spectral changes are predominant. q 2000 Elsevier Science B.V. All rights reserved. Keywords: HrD exchange; Proteins; FTIR correlation spectroscopy
1. Introduction Fourier-transform infrared spectroscopy ŽFTIR. has recently been used for the in situ secondary structure determination of pressurized proteins w1–3x. The development of the two-dimensional Ž2D. correlation spectroscopy w4x opened new possibilities in this field. This new technique is a powerful tool for studying spectral changes induced by external perturbation. Originally the method was developed for sinusoidally varying external perturbations like mechanical stress in polymers. The 2D correlation spec-
) Corresponding author. Fax: q36-1-266-6656; e-mail: [email protected]
troscopy was later generalized by its developer Noda w5x for nonsinusoidal and even for nonperiodic perturbations. 2D IR analysis was used to study the melting of nylon 12 w6x, the solvent-induced perturbation of protein conformation w7x, temperature-induced protein unfolding w8x, but pressure-induced unfolding of proteins is quite new in this line w9x. One can extract additional information, from a 2D analysis of vibrational spectra, because it reflects the response of the system to the external perturbation. Correlation between changes at different spectral positions can be obtained. If overlapping subcomponents of a complex band reply to the external perturbation in different ways, resolution enhancement can also be achieved. These features allow a detailed investigation of the effect of the perturbing parame-
0924-2031r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 4 - 2 0 3 1 Ž 9 9 . 0 0 0 3 7 - 5
120
L. Smeller et al.r Vibrational Spectroscopy 22 (2000) 119–125
ter on the inter- as well as on the intra-molecular interactions. We apply the method to separate overlapping spectral effects due to hydrogen–deuterium ŽHrD. exchange and to changes in the protein backbone conformation. Proteins are stable in their native structure only in very limited temperature and pressure range w10x. According to a general thermodynamic theory, this range is an elliptic area on the p–T space w11,12x. This means that in addition to the well-known heat denaturation, proteins can be unfolded by subjecting them to pressure or cooling. The difference between the effects of high pressure, high temperature and low temperature on the protein structure has been investigated by NMR w13x and UV difference spectroscopy w14x. Nevertheless, there remains the need to follow the changes in secondary structure of proteins under these conditions. The only approach that is available at present is vibrational spectroscopy w10x. In this paper, we used high pressure as perturbing tool for the 2D FTIR spectroscopy in combination with the high pressure diamond anvil cell. The method is demonstrated here using soybean lipoxygenase. Lipoxygenase is a large single-chain protein composed of 839 amino acids. Its structure is composed of two domains: the N-terminal b-barrel is associated with one helix and the major domain is composed of 22 helices and 8 b-strands w15x. The crystal structure of soybean lipoxygenase L-1 is available at 1.4 A resolution w16x. The pressure and temperature inactivation of this enzyme is also of considerable practical interest in view of its role in the quality preservation of food products w17,18x.
2. Experimental Preparation of soybean lipoxygenase is described elsewhere w19x. The protein was dissolved in 10 mM Tris at pD 7.8 in the solvent D 2 O using a protein concentration of 30 mgrml. The solution was left overnight at room temperature and ambient pressure to allow the HrD exchange of the easily accessible and fast exchanging protons. In order to study the kinetics of the slow exchanging protons the pressure cell was thermostated Žafter filling it and applying a
very low pressure to avoid leakage.. Once the chosen temperature was obtained, the sample was pressurized. This was usually done within 4 min. Kinetic measurements were started when the desired pressure was reached. The spectra used for the 2D analysis were taken during a period of 2 h. Infrared spectra were recorded with a Bruker IFS66 FTIR spectrometer equipped with a broadband MCT detector. A total of 250 interferograms were coadded after registration at a resolution of 2 cmy1 . High pressure was generated in a diamond anvil cell ŽDiacell Products, Leicester, UK. and the temperature was measured by a thermocouple. The gasket between the diamonds had an original thickness of 75 mm, which resulted in a sample thickness of approximately 50 mm. The pressure was determined by an internal calibrant, BaSO4 , using the shift of the 983 cmy1 peak w20x. The 2D correlation spectra were calculated by software developed in our laboratory, using the equations of Noda w5x. The reference spectrum is the average for each of the three pressure regions discussed below. For the lower and higher pressure regions a total of eight spectra were used to calculate the correlation spectra. Sixteen spectra were used for the transition region. The spectra were taken in almost equidistant pressure steps of 0.3 " 0.1 kbar. No spectral manipulations Žbackground substraction or resolution enhancement. were performed prior to the 2D analysis. For the representation of the 2D correlation spectra we use the following convention. In a synchronous spectrum, intensity changes observed at two spectral coordinates in opposite direction are indicated by shaded negative crosspeaks. Positive crosspeaks without shading indicate changes in the same direction. In the asynchronous spectrum, the negative peaks are shaded. The interpretation of the sign of the spectral peaks in the asynchronous spectrum is more complex. If a spectral peak at n 1 , n 2 wavenumbers has a positive sign, the spectral changes at n 1 precede the intensity changes at the wavenumber n 2 . This rule is however reversed, if the synchronous plot at the corresponding n 1 , n 2 position has a negative value. The notation of n 1 and n 2 follows the equations of Noda w4,5x. In the standard 2D plots n 1 is on the horizontal axis and n 2 on the vertical one.
L. Smeller et al.r Vibrational Spectroscopy 22 (2000) 119–125
3. Results and discussion Fig. 1 shows the amide IrIX , II and IIX regions of the FTIR spectra in the pressure range of 1–11 kbar. I and II refer to the protonated while IX and IIX refer to the deuterated amide group. The amide I vibration is mainly a C5O stretching, with some small contribution from the N–H bending. It appears between 1600 and 1700 cmy1 , depending on the actual conformation of the protein. In a folded protein the oxygen atom is hydrogen-bonded to the other parts of the polypeptide chain. Therefore, the amide I vibration is sensitive to the secondary structure. Subcomponents of this band can be assigned to different secondary structures w21x, while similar assignments for the amide II Žand IIX . vibrations are less clear-cut. The amide II Žand IIX . modes are mixed vibration modes involving N–H ŽN–D. bending and C–N stretching. Since this vibration strongly involves the hydrogen Žor deuterium. atom the band shifts ca. 100 wavenumbers down upon deuteration w22x. Its position in the protonated protein is at about 1550 cmy1 . The exact position depends only slightly on the specific secondary structure. If the wavenumber maximum of the amide I band is plotted vs. pressure, a sigmoid curve is obtained
X
X
121
with the transition midpoint at 6.5 kbar which can be treated as the denaturation pressure w19x. The wavenumber shift alone, however, is very misleading in this case, because it is not a true shift, but an apparent one, resulting from the relative intensity changes of the subcomponents of the broad amide I band. Analysis of the band with curve fitting is problematic as part of the shifts is due to HrD exchange. Since the spectra do not show very marked changes, we turn to 2D analysis of the data, to gain more detailed information about the conformational change, the HrD exchange and the possible coupling between these processes. The pressure range was divided into three parts for the 2D correlation analysis: low pressure range Ž2–4 kbar., the transition region Ž4.7–9.0 kbar., and the upper pressure region Ž8.5–10.6 kbar.. Fig. 2 shows the synchronous and asynchronous spectra of lipoxygenase for the low pressure range. The synchronous spectrum in the low pressure region showed intense auto and crosspeaks in the amide IIX region Ž1472, 1391 cmy1 . while very minor changes can be seen in the amide I band. The peaks of amide IIX region are very sensitive to HrD exchange. However, since there are no negative
Fig. 1. Amide IrI , II and II regions of the FTIR spectra of soybean lipoxygenase in the pressure range of 1–11 kbar. Pressure increases from bottom to top in equal pressurertime steps.
122
L. Smeller et al.r Vibrational Spectroscopy 22 (2000) 119–125
Fig. 2. Synchronous Ža. and asynchronous Žb. spectra of lipoxygenase for the low pressure range Ž2.3–4.4 kbar.. The negative peaks are shaded. The reference spectrum can be seen on the side of the 2D plot.
crosspeaks for the amide II and amide IIX bands, the peaks at 1472 and 1391 cmy1 can also reflect the slight elastic deformation of the protein. Unfortunately, the assignment of the components of the amide IIX band to the different secondary structures is not as simple as for the amide I band. Therefore, we cannot assign these changes to any part of structural component of the protein. The small autopeak at 1620 cmy1 might indicate the increase of the intermolecular interactions, while the negative crosspeak at 1472 and 1655 cmy1 indicates some loss in the helical structure. It is of interest to note that the active site is located in the domain that contains all the helices w16x. The asynchronous plot indicates that the spectral changes at 1620 cmy1 precede the amide IIX changes. The 2D spectra obtained in the transition region Ž4.5–9 kbar. can be seen on Fig. 3. The synchronous spectrum is much better populated by correlation peaks than the former plot was. Conformational changes and exchange effects are both present in these spectra. The following spectral bands show remarkable contribution to the 2D spectra: 1686, 1652, 1619, 1541, 1475, 1424 and 1365 cmy1 . The
spectral changes in the amide I region reflect the unfolding of the protein. The decrease of the folded structure can be seen from the peak at 1655, which is anticorrelated to the increasing amide IIX band and positively correlated to the diminishing amide II band. The anticorrelation between the 1655 and 1620 bands might indicate further increasing intermolecular interactions. The amide II and IIX bands anticorrelate as one would expect. The analysis of the asynchronous spectrum results in the following sequence of intensity changes of the main peaks: 1620 cmy1 Žamide IrIX . 1473 cmy1 Žamide IIX . 1650 cmy1 Žamide IrIX . 1540 cmy1 Žamide II.. The other peaks change in a manner uncorrelated with the above ones. This sequence shows that the conformational changes of the protein are correlated with the HrD exchange. Loosening of the original conformation enhances the exchange, while the exchanged protein undergoes further unfolding and loss of secondary structure. The pressure region where the protein is already unfolded gives the 2D spectra which can be seen in Fig. 4. Above 8.5 kbar the protein lost its enzymatic function completely, but fragments of the secondary
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™™
L. Smeller et al.r Vibrational Spectroscopy 22 (2000) 119–125
123
Fig. 3. Synchronous Ža. and asynchronous Žb. spectra of lipoxygenase for the transition regime Ž4.7–9.0 kbar.. The negative peaks are shaded. The reference spectrum can be seen on the side of the 2D plot.
structure are still present. This can be seen from the autopeak in the synchronous spectrum at 1640 cmy1
which reflects the further increase of the unordered chain. The remaining ordered structure of the protein
Fig. 4. Synchronous Ža. and asynchronous Žb. spectra of lipoxygenase in the high pressure region Ž8.5–10.6 kbar.. The negative peaks are shaded. The reference spectrum can be seen on the side of the 2D plot.
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becomes smaller by further increasing the pressure in this high pressure range. Consequently the less accessible protons will also exchange. This can be seen in the amide II and IIX region by observing the auto and crosspeaks at 1380, 1460, 1525 and 1550 cmy1 . Since there is again a band in the amide II Ž1525 cmy1 . which is not anticorrelated with the amide IIX ones, we assign it to the conformational changes in the folded fragments, instead of the HrD exchange. In the asynchronous spectrum we note that the amide II and IIX changes Žat 1380, 1460, 1525 cmy1 . follow the weak spectral changes at 1640 cmy1 associated to the unordered structure. From the previous analysis, it can be noted that the position of the correlation peaks for the same band differs slightly for the different pressure regions. This may be attributed to pressure-induced frequency shifts, pressure-induced distortions of the secondary structural elements of the protein or a combination of both effects w23x. In order to clarify further the connection between the exchange and conformational changes, kinetic experiments were performed slightly below and above the transition pressure Ž6.5 kbar.. Since the fast accessible protons were already exchanged, Žsee Section 2 for the details of the kinetic measurements., the exchange observed after the pressure jump can be attributed to the protons that are exposed to the solvent due to the pressure induced conformational alterations in the protein structure. Fig. 5 shows the synchronous spectra obtained at Ža. 5.2 and Žb. 7.3 kbar. After a pressure jump to 5.2 kbar, the main peak can be seen in the amide I region. This means that some conformational changes started already at this pressure. Small crosspeaks at 1650, 1475 cmy1 and at 1650, 1545 cmy1 indicate the small exchange induced by the loosening of the conformation. Since the amide IIrIIX bands are very sensitive to the HrD exchange, the small intensity of the peaks indicates that the number of protons that are exposed to the D 2 O solvent due to the jump to 5.2 kbar is very small. This means that the conformation of the protein did not change considerably, and only a few hydrogen atoms became exposed to the solvent. Fig. 5b shows the synchronous spectrum of the kinetic experiment at 7.2 kbar. The fact that the amide II and IIX auto- and crosspeaks dominate the spectrum, shows that the number of protons accessi-
Fig. 5. Synchronous spectra of kinetic experiments on lipoxygenase at high pressure Ža.: at 5.2 kbar, Žb. at 7.3 kbar. The negative peaks are shaded. The reference spectrum can be seen on the side of the 2D plot.
ble to the solvent is increased by pressure-induced unfolding of the protein. The absolute intensities of the amide II and IIX autopeaks are five times higher on this picture compared to the 5.2 kbar one, where the autopeaks are not visible due to their very low intensity. Since the kinetic experiment was started
L. Smeller et al.r Vibrational Spectroscopy 22 (2000) 119–125
after reaching the desired pressure, the relative small intensity of the amide I peak suggests, that the unfolding of the protein is a fast process, the equilibrium conformation was reached in 3–4 min. This was the time required for increasing the pressure to the desired value. The conformational changes were completed before the start of the kinetic experiment, that is why the 2D spectrum mainly reflects the slower HrD exchange process induced by unfolding. It is of interest to compare the differences between a small protein, such as bovine pancreatic trypsin inhibitor ŽBPTI, 8 kDa. and lipoxygenase, which is a large protein Ž102 kDa.. For BPTI we observe an increased rate of HrD exchange below 5 kbar w2x. Above that pressure only a conformational change takes place. For BPTI it seems that the low pressure regime acts preferentially on the conformational dynamics thus promoting the HrD exchange. In the case of lipoxygenase, pressure induces irreversible conformational changes resulting in an increased exposure of interior parts of the protein to the solvent. Summarizing, we can conclude that the application of pressure as a perturbing parameter to 2D FTIR gives new information about the pressure effect on the structure and dynamics of macromolecular systems. Application of this analysis to biological systems like proteins can unravel complex phenomena like the relation of conformational changes and HrD exchange processes. These effects are difficult to separate in the conventional one-dimensional spectra. Overlapping bands of amide I region can be separated under these conditions when they respond to the perturbation in different ways.
Acknowledgements This research was supported in part by an EU project ŽFAIR CT96-1175., FWO, Flanders, the
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