Two-Dimensional Infrared Spectroscopy and Principle Component

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J. Phys. Chem. B 2001, 105, 6251-6259

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Two-Dimensional Infrared Spectroscopy and Principle Component Analysis Studies of the Secondary Structure and Kinetics of Hydrogen-Deuterium Exchange of Human Serum Albumin Yuqing Wu,†,‡ Koichi Murayama,† and Yukihiro Ozaki*,† Department of Chemistry, School of Science, Kwansei-Gakuin UniVersity, Nishinomiya 662-8501, Japan, and Key Laboratory for Supramolecular Structure and Spectroscopy of Ministry of Education, Jilin UniVersity, 130023, People’s Republic of China ReceiVed: January 22, 2001; In Final Form: April 9, 2001

Infrared (IR) spectra were measured as a function of time after dissolving human serum albumin (HSA) into D2O to investigate the secondary structure and the kinetics of hydrogen-deuterium (H/D) exchange. Twodimensional (2D) IR correlation spectroscopy and principal component analysis (PCA) were used to analyze the obtained spectra. Two-dimensional IR spectra in the amide I and amide II (and II′) regions were generated from time-dependent spectral variations in different exposure time domains of HSA in the D2O solution. The synchronous and asynchronous spectra in each time domain provide a clear separation of amide bands due to the different secondary structures and the asynchronous spectra show the specific sequence of the secondary structure exposed to the H/D exchange. PCA was used to select the appropriate time domains for the calculation of 2D correlation spectra. It was found that the loadings plots of PCA are useful also to assist the band assignments in the amide I and II region and to investigate the mechanism of the H/D exchange. The present study demonstrates the potential of PCA-2D correlation analysis combined method in the efficient separation of component bands due to different secondary structures and in the study of the kinetics of H/D exchange reaction.

Introduction Fourier transform infrared (FT-IR) spectroscopy has emerged as a powerful technique for qualitative and quantitative estimation of the secondary structures of proteins.1-5 By using FT-IR one can investigate the secondary structures even for proteins with huge molecular weight. In the IR region, the frequencies of bands due to the amide I, II, and III vibrations are sensitive to the secondary structure elements of proteins. The separation of broad amide I band into component bands and their assignment to various secondary structures, such as R-helix, β-sheet, β-turn, and random coil have been the subjects of numerous studies.1-5 Usually, second derivative, Fourier selfdeconvolution (FSD) and curve fitting are used to deconvolute the amide I band. However, the results obtained by the above methods are not always conclusive, especially for complex proteins consisting of various secondary structures. To unravel the highly overlapped spectra in the amide I and amide II region, hydrogen-deuterium (H/D) exchange of the amide protons is also useful.6-13 Since the amide protons associated with each conformation are not exchanged simultaneously, the contributions from different secondary structures to the amide bands can be separated by using the H/D exchange. Deuterium exchange rates are also a powerful probe for amide structure, * To whom all correspondence should be sent. Mailing address: Department of Chemistry, School of Science, Kwansei-Gakuin University, Nishinomiya 662-8501, Japan. FAX: +81-798-51-0914. E-mail: ozaki@ kwansei.ac.jp. † Department of Chemistry, Kwansei Gakuin University. ‡ Key Laboratory for Supramolecular Structure and Spectroscopy of Ministry of Education, Jilin University.

providing information about solvent accessibility and hydrogen bond stability of amide protons. In recent years generalized two-dimensional (2D) correlation spectroscopy14 has been introduced as a new-technique for analyzing amide I and amide II bands of proteins.15-18 Twodimensional correlation spectroscopy is very useful for the protein research because of three major reasons. One is that 2D correlation spectroscopy deconvolute amide bands into component bands due to different secondary structure. Another is that it enables one to establish the correlation between different secondary structures of protein through selective correlation peaks for given perturbation. Yet another is that it gives information about specific order of secondary structural changes and changes in side chains under various environments. The purpose of the present study is to show the usefulness of principal component analysis (PCA)-2D correlation analysis combined method in analyzing time-dependent changes in IR spectra of human serum albumin (HSA) caused by the H/D exchange. PCA was used to divide the spectra into several deuterium exposure time domains. Two-dimensional correlation analysis was applied to several spectra in each time domain to enhance the spectral resolution in the amide I and II regions. Time after dissolving HSA into D2O was used as the external perturbation. The 2D correlation analysis has provided deeper insight into the mechanism of the H/D exchange in the amide protons of different secondary structures. Nabet and Pezolet15 reported 2D IR correlation spectroscopy study on the H/D exchange of myoglobin (Mb) earlier. They showed that the use of two different exchange time domains is very efficient to separate the fast kinetics from the slow ones.15 The present

10.1021/jp010225b CCC: $20.00 © 2001 American Chemical Society Published on Web 06/07/2001

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study has two significant novelty compared with their study. One is that we use PCA. PCA is powerful not only in selecting the time domains but also in the band assignments and the study of the kinetics of the H/D exchange. Loadings plots play very important roles in the later two points. Another is that we analyze asynchronous spectra in more detail. Human serum albumin (HSA) is one of the most important proteins in human blood.19-27 It is composed of three domains, I, IIA, IIB, and III by 585 amino acids, and is made up of 98 acidic side chains and 100 basic side chains. HSA molecule has 67% helix, no β-sheet, 10% turn, and 23% extended chain in a crystalline state.21-23 It is well-known that the three domains have different stability against external perturbation and that the R-helix structures in HSA are considerably stable with temperature and pH changes.19-21 The present study provides new insight into the following three points: (i) the potential of the combination of PCA and 2D IR correlation analysis in this field; (ii) the band assignments in the amide I and amide II region for the complicated different secondary structures in HSA; (iii) the kinetics of HSA during the H/D exchange. Experimental Section 1. Materials. HSA (Grade V) was purchased from Sigma Chemical Corporation and used without further purification. The purity of HSA was proved by electrophoresis to be approximately 96-99%. Deuterium oxide (D2O) was obtained from Sigma Chemical Corporation. The minimum isotopic purity is 99.996 atom % D. 2. Hydrogen/Deuterium Exchange and IR Spectroscopy. Two milligrams of HSA was dissolved in 100 µL of D2O. The moment of dissolving the protein into D2O was defined as the initiation of the H/D exchange (t ) 0). Next, the sample solution was introduced by a microsyringe into a CaF2 cell with a thickness of 16 µm. IR spectra were measured at a 2 cm-1 spectral resolution with a Nicolet Magna 760 FT-IR/NIR spectrometer equipped with a liquid nitrogen cooled mercury cadmium telluride (MCT) detector. During the first 5 min of the H/D exchange a spectrum was obtained every 30s as an average of 16 scans. After that, the time interval was increased exponentially; the final spectrum was taken 5 h after the initiation of the exchange. 3. Analysis of Infrared Spectra. IR spectra of protein-free sample was also measured under the same experimental conditions as above. The spectrum of the protein-free sample corresponding to the same time point was subtracted from the spectrum of HSA in D2O. The obtained spectra were corrected for the contributions from atmospheric water and subsequently smoothed with a seven-point Savitsky-Golay function to reduce the noise. Second derivative of the IR spectra were calculated with an 11-point Savitsky-Golay function. For all the data presented in Figure 2, the areas of amide II and amide II′ bands were normalized to that of amide I band to eliminate possible fluctuations in intensity induced by the instrument. The spectral range for the area integration for the amide I, amide II, and amide II′ bands are 1700-1601, 1598-1505, and 1495-1375 cm-1, respectively. 6,11,13 4. Principal Component Analysis (PCA). PCA is a wellknown pattern recognition and multivariate data display method.28-30 It can compare not only any object clusters, but also display any relationships among variables as well as variables and objects. Prior to PCA, the pretreatment of the data, such as raw centering and standardization was manipulated using Excel 5.0 spreadsheet. This scaling procedure ensures that all

Figure 1. (A) An FT-IR spectrum of HSA in a H2O solution and those of HSA in a D2O solution as a function of exposure time. The spectrum with the dashed line: the H2O solution. The spectra with solid line: the D2O solution measured (from top to bottom) from 1.5 to 260 min after the initiation of the exposure of HSA to D2O at 25 °C. In each spectrum the spectrum of H2O or D2O was subtracted. (B) A second derivative of the spectrum of HSA in the H2O solution (dash-dot line) and those measured at 1.5 min (dashed line) and at 260 min (solid line) after the initiation of the H/D exchange of HSA in the D2O solution, at 25 °C.

the variables have the same weighting in the PC model. After the pretreatment, the appropriately formatted data were exported to Unscrambler 6.0 for the PCA analysis. 5. Two-Dimensional Correlation Analysis. We employed software named KG/2D for the 2D correlation analysis composed by Y. Wang (Kwansei Gakuin University) with the Array Basic programming language (The Galactic Industries Corp.).31 This 2D software was programmed on the basis of the developed algorithm of generalized 2D correlation spectroscopy.32 Results 1. Hydrogen-Deuterium Exchanges in Amide Protons of HSA Monitored by IR Spectra. Figure 1A shows a series of IR spectra measured as a function of time after dissolving HSA into D2O (pD 7.0, pD ) pDread + 0.4433). The spectrum shown by a broken line is the spectrum of HSA in H2O. The amide I region of the spectra (1700-1600 cm-1) shows a broad maximum at 1654 cm-1 characteristic of a R-helical structure, in a good agreement with the results of X-ray crystallography23,24,34 and the previous IR studies of HSA in H2O solution.27,35-37 The IR spectra measured over 4 h after the

Hydrogen-Deuterium Exchange of Human Serum Albumin

J. Phys. Chem. B, Vol. 105, No. 26, 2001 6253 at 1585, 1565, 1550, and 1515 cm-1 in the amide II region, and at 1469, 1457, 1438, 1422, and 1402 cm-1 in amide II′ region. The bands at 1550 and 1515 cm-1 are attributed to the R-helices38,39 and tyrosine side chains,25 respectively. A band near 1585 cm-1 may arise from the carboxylate groups of two amino acid residues: glutamic acid and aspartic acid.25 The bands between 1469 and 1438 cm-1 are assigned to the deuterated amide II groups (amide II′) overlapped with a band arising from the bending mode of HOD.6-9,25 We monitor the reaction of -CONH- + D2O f -COND+ HOD by measuring the decrease in the intensity of the amide II region. During the H/D exchange the intensity of the area of amide I band in the 1700-1600 cm-1 region remains practically constant. Therefore, we take the ratio of the area of the amide II band (or amide II′) to that of the amide I band centered at 1654 cm-1 (Aamide II /Aamide I or Aamide II′ /Aamide I) to eliminate possible fluctuations in intensity (see the experiment part).38 To estimate the fraction of the nonexchanged amide protons (X), the initial value of Aamide II/Aamide I is expressed by the ratio of the area of the amide II band to that of amide I band of HSA in the H2O solution.35,36 It is found that the initial value of Aamide II/ Aamide I is 0.56, which is somewhat larger than that for HSA in a H2O solution at pH 7.0 obtained by Hvidt and Wallevik.26 The difference between our result and their result may arise from the slight difference in the pH value (pH 6.6, pH 7.0) and/ or in the small bias resulting from the subtraction of the water spectrum from the protein spectrum. Figure 2A plots the ratios of Aamide II/Aamide I and Aamide II′/ Aamide I as a function of the time. The decrease in the Aamide II/Aamide I may be ascribed as the sum of the individual exponential decay for each amide proton. Apparently, a large number of amide protons remain unchanged even after a few hours. The fraction, X, of the nonexchanged amide protons at a time, t, can be estimated as39

X ) Aamide II/0.56Aamide I Figure 2. (A) The ratios of the area of amide II or amide II′ divided by that of amide I for the spectra shown in Figure 1A vs time after the initiation of the H/D exchange in HSA in the D2O solution. (B) Fraction of the nonexchanged amide protons in HSA in the D2O solution.

exposure of HSA to D2O exhibit a number of spectral changes due to the H/D exchanges in the amide N-H groups. The most noticeable change in Figure 1A is a dramatic decrease in the intensity of the amide II band (primarily coupling of the N-H bending and C-N stretching modes) centered at 1552 cm-1 and a concomitant intensity increase at 1450 cm-1 (amide II′; coupling of the N-D bending and C-N stretching modes). The intensity decrease in the amide II band provides a direct measure for both the time course and the extent of amide H/D exchange (see Figure 2). To resolve the overlapping band components under the amide I contour and to explore kinetics of the H/D exchange, a series of the second derivative spectra were calculated and three of them are shown in Figure 1B. The spectrum with the dash-dot line correspond to HSA in the H2O solution, and the spectra with the dashed and connected lines are those measured 1.5 and 260 min after the start of the H/D exchange, respectively. Frequency shifts for the R-helix band at 1656 cm-1 and the β-strands band at 1630 cm-1 are very small, but a significant shift is observed for a band near 1686 cm-1 due to the β-turn structures. These results suggest that the H/D exchange does not change much the conformation of R-helices and the β-strands. In Figure 1B, two groups of bands are deconvoluted

Figure 2B plots the fraction of X as the function of time. It can be seen from Figure 2B that roughly 50% of the amide protons undergo the H/D exchange within 2 min after the exposure of HSA to D2O, an additional 12% over the next 30 min, and a further 10% over the next a few hours. Roughly 25% of the amide protons resist to being deuterated even 4 h after the exposure of HSA to D2O at 25 °C. The plots in Figure 2A,B show the H/D exchange rate as a function of time. However, the mechanism of the exchange is still unclear from the plots. Under constant experimental conditions (pH and temperature), the rate of H/D exchange and the extent of the exchange at a given time period largely depend on the structural mobility of proteins; the more mobile (less rigid) the protein structure, the faster the rate of H/D exchange and the greater the final equilibrium level of exchange.40,41 We have to explore what kinds of secondary structural elements are involved in the fast exchange, and which parts are involved beyond this time frame or resist the exchange. Two-dimensional correlation analysis and PCA will give evidential answer for such questions. 2. PCA Study. Recently, Czarnik-Matusewicz et al.42 used PCA to separate temperature-dependent IR spectra of cottoncellulose into several groups before the calculation of 2D correlation spectra. We employ PCA not only to separate the IR spectra of HSA in the D2O solution into different groups but also to deconvolute amide I and amide II bands and to investigate the kinetics of HSA during the H/D exchange. Figure

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Figure 3. A PC1 vs PC2 plot of PCA for the time-dependent IR spectra of HSA shown in Figure 1A.

3 shows a two-factor score plot of PCA factor 1 (PC1) and factor 2 (PC2) for the time-dependent IR spectra in the 17201360 cm-1 region. The two PCs account for nearly 99.9% of the total variance. One can easily find three groups, groups I, II, and III in the score plot. Groups I, II, and III consist of the spectra measured between 1.8 (HD02) and 5.3 (HD06) min, between 6.4 (HD07) and 13.4 (HD16) min, and between 25.2 (HD19) and 181 (HD27) min after the initiation of the H/D exposure, respectively. The result in Figure 3 provides very important basis for the selection of the time domains for the 2D correlation analysis. Figure 4 A,B depict loadings plots (or abstract spectra) of PC1 and PC2 for the score plot in Figure 3, respectively. The loadings plot of PC1 shows positive peaks at 1682, 1667, 1663, and 1540 cm-1, which mainly correspond to the bands due to β-turns (bands at 1682, 1667, and 1663 cm-1 in the amide I region)25 and β-strands (a band at 1540 cm-1 in the amide II region).38,39 This means that the exchanges in the amide groups of β-turns dominate the exchanges in groups I and II, which both have the positive PC1 values in Figure 3. The negative peaks at 1625 cm-1, and 1452 and 1437 cm-1 may be ascribed to the amide I band of intermolecular β-strands and the amide II′ bands of R-helices overlapped with the band arises from the bending mode of HOD, respectively.43,44 The negative peaks in PC1 reflect the opposite change direction of these features. It seems that the formation of the intermolecular β-strands, HOD, and the deuterated R-helices are predominant in group III (Figure 3). The loadings plot of PC2 shows all positive bands at 1698, 1687, 1672, 1666, 1650, 1630, and 1620 cm-1 in the amide I region, those at 1573, 1558, and 1538 cm-1 in the amide II region, and those at 1469 and 1460 cm-1 in the amide II′ region. These positive peaks mean that groups I and III are characterized

by these band features. They contribute greatly to the separation of groups I and III from group II, especially the separation between groups I and II. Even though the characteristic features of groups I and II are the H/D exchanges in the β-turns, there are still great differences between them as revealed by the positive peaks in the loadings plot for PC2. The loadings plot for PC2 suggest that the H/D exchanges in the R-helices (1650 and 1558 cm-1) and the formation of aggregated β-strands25 (between 1630 and 1620 cm-1; band assignments in this region will be discussed in detail in the next part) dominate the changes in group III. The above peaks contribute greatly to the separation of group III from group II. This kind of characterization and the differentiation should also be revealed by the asynchronous 2D correlation spectra constructed from the different exchange time domains. 3. Two-Dimensional IR Correlation Spectroscopy. Figure 5 shows the (a) synchronous and (b) asynchronous correlation maps of HSA for the rapidly exchange amide protons, which are constructed from the five spectra in group I (in Figure 3). The power spectrum along the diagonal line of the synchronous spectrum is shown in Figure 6 I. It shows that the synchronous map is characterized by autopeaks at 1683, 1666, and 1625 cm-1 in the amide I region, the autopeak at 1542 cm-1 in the amide II region, and the autopeaks at 1452 and 1437 cm-1 in the amide II′ region. The appearance of these autopeaks mean that the intensities of these amide bands change significantly with the progress of the H/D exchange. As already discussed, the amide I components at 1683 and 1666 cm-1 are assigned to the β-turn structures.35,36 Therefore, it seems that the amide groups associated with the β-turn structures are involved in the first H/D exchange during the deuteration process. It is noted that in the synchronous map in Figure 5A that the peak at 1625 cm-1 shares a negative cross-peak with the bands

Hydrogen-Deuterium Exchange of Human Serum Albumin

Figure 4. Loadings plots of PC1 (A) and PC2 (B) for the score plot in Figure 3.

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Figure 7. Slice spectra at 1543 cm-1 extracted from the asynchronous 2D contour maps shown in Figures 5B, 8B, and 9 (B); (I) from Figure 5B, group I; (II) from Figure 8B, groups II and III from Figure 9B, group III.

Figure 5. (A) Synchronous and (B) Asynchronous 2D IR correlation spectra of HSA calculated from five spectra (group I), which measured between 1.8 and 5.3 min after the initiation of the H/D exchange.

Figure 6. Power spectra extracted from the synchronous 2D contour maps shown in Figures 5A, 8A and 9A; (I) from Figure 5A, group I; (II) from Figure 8A, group II; and (III) from Figure 9A, group III. (I′) A slice spectrum at 1667 cm-1 extracted from the synchronous 2D contour maps shown in Figure 5A.

in the 1690-1660 cm-1 region and those in the amide II region. Therefore, it is very likely that the band at 1625 cm-1 is associated with the H/D exchange of amide groups. Usually, the amide I band near 1625 cm-1 is assigned to a β-sheet structure. However, for Mb15 and HSA,35,36 which do not have a β-sheet structure, the band near 1625 cm-1 may assigned to intermolecular β-strands (or sheets) structure.43,44 There is an

increase of band at 1625 cm-1 because the increase of intermolecular with the deuterium time. 25,43,44 The strong autopeaks is observed at 1542 cm-1 in the amide II region, since the amide II vibrations are much more strongly affected by the deuteration of amide groups than the amide I vibrations.40 The peak at 1542 cm-1 is correlated with all the three bands in the amide I region, indicating that the changes embedded in the broad band centered at 1542 cm-1 are attributed to the same secondary structural elements as those observed in the amide I region. In the amide II′ region, two autopeaks are observed at 1452 and 1437 cm-1, which may be ascribed to different secondary structures of the deuterated amide II (i.e., amide II′) bands.40 The peak at 1452 cm-1 shares a cross-peak with the peaks at 1683, 1666, 1625, and 1542 cm-1, indicating that the broad feature centered at 1452 cm-1 contains contributions from the deuterated β-turns and β-strands structures. Unlike the synchronous map in the amide I region, those in the amide II and amide II′ regions do not display highly resolved spectral contributions from the different secondary structures. It is of particular note that the slice spectrum at 1666 cm-1 extracted from the synchronous spectrum in Figure 5A (Figure 6I′) is very similar to the loadings plot for PC1 in Figure 4A. The results of the 2D correlation analysis and PCA are in a very good agreement. The asynchronous correlation spectrum in Figure 5B yields much better deconvolution of the spectral bands in the amide II and amide II′ regions. The asynchronous spectrum looks complicated, but the positions of deconvoluted peak in the asynchronous spectrum are in a good agreement with those in the second derivative spectra (Figures 1B and 5B). Figure 7I depict a slice spectrum extracted at 1543 cm-1 from the asynchronous spectrum in Figure 5B. The deconvolution of amide bands due to different secondary structures is demonstrated more clearly in Figure 7I. The slice spectrum in Figure 7I shows four new bands at 1655, 1640, 1613, and 1604 cm-1 in the amide I region, assigned to R-helices (1655 cm-1), intramolecular β-strands or random coil (1640 cm-1), and side chains (1613 and 1604 cm-1), respectively.35,36 In fact, the H/D exchanges in the side chains should occur very fast according to the proposed two crucial inhibition factor for the H/D exchange in native proteins.25,44,45 However, the bands at 1613 and 1604 cm-1 arising from the tyrosine side chains are not observed in the synchronous spectrum constructed from group I (Figures 5A and 6 I). Probably, those are overlapped by the

Hydrogen-Deuterium Exchange of Human Serum Albumin strong band at 1625 cm-1. Usually, the band at 1640 cm-1 is attributed to a β-sheet or random coil structure for proteins.1-6 For HSA it may be assigned to intramolecular β-strands or random coil.25,35,36 This band is not observed in the second derivative spectrum of HSA in the H2O solution (Figure 2, dotted line). However, note that a very weak feature is observed at 1640 cm-1 in the second derivative spectrum of HSA in the D2O solution (Figure 2, dashed and connected lines). Its subtle change due to the H/D exchange cannot be detected in the synchronous 2D contour map. In contrast, the asynchronous 2D correlation spectrum demonstrates its strong deconvolution power. Several new bands are observed at 1591, 1578, 1555, and 1533 cm-1 in the amid II region and at 1486 and 1421 cm-1 in the amide II′ region in Figure 7I. These bands can be assigned to side chains (1591 cm-1),25 β-turns (1578 and 1486 cm-1), R-helices (1555 cm-1), and β-strands (1533 and 1421 cm-1), respectively.38,39 In a 2D asynchronous correlation spectrum, cross-peaks between two spectral features are developed only when their intensities vary out-of-phase with each other.32 For a study on the H/D exchange process of a protein, the asynchronous map allows one to explore the order in the H/D exchange of secondary structure elements. This may not be achieved by the conventional analysis of intensity changes in IR spectra of proteins. In Figure 5B, asynchronous cross-peaks are observed at (1640 and 1669) and (1640 and 1685) cm-1 in the amide I region. However, no asynchronous cross-peak is developed among the bands at 1685, 1669, and 1625 cm-1, suggesting that the secondary structures giving the peaks at 1685, 1669, and 1625 cm-1 show very similar or almost equal H/D exchange kinetics. In contrast, it seems that the secondary structure that has the band at 1640 cm-1 shows quite different behavior in the H/D exchange from those with the amide I band at 1685 and 1669 cm-1. On the basis of the rule for an asynchronous spectrum (Ψ(ν1,ν2)) proposed by Noda,14 one can investigate the specific order of the spectral intensity changes occurring during the measurement. If the sign of an asynchronous cross-peak (Ψ(ν1,ν2)) on the left up side of the asynchronous spectrum is positive, the intensity change at ν1 takes place predominantly before that at ν2. On the other hand, if the change occurs after ν2, the sign becomes negative. It must be kept in mind that this rule is reversed if the corresponding synchronous spectrum (Φ(ν1,ν2)) shows negative sign. The signs of cross-peaks at (1640 and 1669) and (1640 and 1685) cm-1 suggest that the H/D exchange occurs in the β-strands (1640 cm-1) earlier than in the β-turn structures (1685 and 1669 cm-1); that is,

β-strands (1640 cm-1) > β-turns (1685 and 1669 cm-1) Figure 8 shows the (A) synchronous and (B) asynchronous correlation maps of HSA calculated from nine IR spectra (spectra from HD07 to HD16, group II in Figure 3) measured between 6.4 and 13.4 min after the initiation of the H/D exchange. Figures 6II and 7II show the power spectrum and the slice spectrum extracted from the synchronous and asynchronous spectra in Figure 8A,B, respectively. The synchronous map in Figure 8A looks similar to that in Figure 5A generated from the five spectra in group I. However, close inspection of the two power spectra (Figure 6I,II) calculated from the synchronous maps in Figures 5A and 8A, shows significant differences between them. The most notable difference is the appearance of an autopeak near 1658 cm-1 in Figure 6II. This band obviously arises from the R-helix structure. This observa-

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Figure 8. (A) Synchronous and (B) asynchronous 2D IR correlation spectra of HSA calculated from nine spectra (group II) measured between 6.4 and 13.4 min after the initiation of the H/D exchange.

tion reveals that besides the H/D exchanges in the β-turns (1678 cm-1), the D2O molecules start going into the accessible parts of R-helices in the second time domain of the H/D exchange. One more notable differences between the slice spectra in Figure 6I,II is observed in the amide II′ region; the increasing band at 1462 cm-1 may be assigned to the bending mode of HOD.25 The asynchronous contour map in Figure 8B displays the deconvolution of bands corresponding to different secondary structure elements. The slice spectrum at 1543 cm-1 is shown in Figure 7II. It shows that positive or negative cross-peaks are developed between the 1543 cm-1 band and the amide I bands at 1683, 1671, 1659, 1648, 1637, and 1619 cm-1 in the asynchronous spectrum in Figure 8B. These amide I bands share cross-peaks at (1671 and 1683), (1653 and 1671), (1653 and 1683), (1623 and 1671), and (1623 and 1653) cm-1. The signs of the cross-peaks suggest the following sequences of the H/D exchanges in the amide protons.

β-turns (1683 cm-1) > aggregated β-strands (1623 cm-1) > β-turns (1671 cm-1) > 1653 cm-1(R-helices) Therefore, it seems that in the second time domain of the exchange, the amide protons in another kind of β-turns (1671 cm-1) are involved in the H/D exchanges. They are more accessible to the D2O molecules than the accessible parts of R-helix structures. Figure 9 illustrates the (A) synchronous and (B) asynchronous correlation maps of HSA, constructed from the nine spectra

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β-turns (1663 cm-1) > β-turns (1683 cm-1), random coil (1643, 1540 cm-1) > R-helices (1653, 1549 cm-1) > aggregated β-strand (1623 cm-1) Discussion

Figure 9. (A) Synchronous and (B) asynchronous 2D IR correlation spectra of HSA calculated from nine spectra (group III) measured between 25.2 and 181 min after the initiation of the H/D exchange.

measured between 25.2 and 181 min after the beginning of H/D exchange process (group III). It can be seen from the synchronous map in Figure 9A and a power spectrum shown in Figure 6 (III) that the exchanges in the R-helices (1656 cm-1) and the formation of aggregated β-strands (1623 cm-1) dominate in this time domain. Of note is that the bands at 1656 and 1623 cm-1 share a negative cross-peak at (1656 and 1623) cm-1. This negative cross-peak indicates that the molecular aggregation is still involved in the with the H/D exchange. The sign of the cross-peak at (1623 and 1656) cm-1 in both the synchronous and asynchronous spectra in Figure 9A,B reveals that the H/D exchanges in the R-helices (1656 cm-1) occur before the formation of β-strands (1623 cm-1). A slice spectrum at 1543 cm-1 extracted from the asynchronous spectrum in Figure 9B is shown in Figure 7III. In comparison with the power spectrum in Figure 6III, it demonstrates many deconvoluted bands due to the different secondary structures, especially in the amide I and amide II regions. There is only one broad band centered at 1656 cm-1 in the power spectrum (Figure 6III), whose frequency is slightly higher than that of the general R-helix band (usually around at 1653 cm-1 for HSA). This band is split into two bands at 1663 and 1653 cm-1, which are ascribed to β-turn and R-helix structures, respectively. Another two bands at 1683 and 1643 cm-1 are assigned to β-turns and random coil structures,25 respectively. The sequence order of H/D exchange revealed by the sign of the cross-peaks in the asynchronous spectrum (Figure 9B) is summarized as follows:

1. Hydrogen-Deuterium Exchange Behavior of HSA. In a synchronous map, a positive cross-peak indicates that the intensities of bands at the corresponding frequencies are increasing or decreasing simultaneously, while a negative peak indicates that the band intensities corresponding to these two components vary in opposite directions.14 During the H/D exchange process, bands due to the amide I and amide II vibrations decrease in intensity. The amide I band and corresponding amide I′ band are very close in frequency, while the amide II band and corresponding amide II′ band are separated by about 100 cm-1.43 Therefore, peaks associated with correlations between the amide I and amide II vibrations should be positive and those associated with correlations between the amide I and amide I′ vibrations and between the amide II and amide II′ vibrations should be negative. From this consideration, the peaks appearing at 1683 and 1666, and 1653 cm-1 may be assigned to the amide I bands of the β-turns, and R-helices, respectively, while the band at 1625 cm-1 should be associated with the amide I′ vibration of aggregated β-strands structures. One would expect that at low temperature no intermolecular β-strands is formed. However, the evidence for the aggregation of protein in a D2O solution at 14 °C was reported by Van Stokkum et al. as revealed by the appearance of band at 1625 cm-1.25 2. Kinetics of the H/D Exchange. It is expected that two distinct physical phenomena occurs during hydrogen-deuterium exchange of HSA: one is related to the hydration effect and the other is associated with hydrogen-deuterium exchange. This is especially the case when the spectra are measured just after preparing the D2O solution of HSA. Therefore, we examined if the effect of hydration appeared in the IR spectra. However, we could not find any evidence for the effect of hydration. Probably, the effect is too small to be detected by IR spectroscopy. Two types of inhibition for the H/D exchange in native proteins have been well-known; strong hydrogen bonding in a secondary structure and inaccessibility of the solvent molecules (D2O) to a secondary structure due to tertiary structure.45,46 Accordingly, the amide protons associated with the different secondary structures would not be deuterated at the same rate. Also, the magnitude of the deuteration shift is dependent upon the secondary structures. It is known that the positions of amide I bands associated with random coil and β-sheet structures are more sensitive to the H/D exchange than those associated with R-helix structures. Amide I bands of random coil structures in proteins shift from around 1660-1655 cm-1 to 1645-1640 cm-1.6 Thus, the 1640 cm-1 band might be due to deuterated random coil structures. The 2D IR correlation analysis in the different time domains is employed to clarify the kinetics of the H/D exchange. The power spectra in Figure 6 extracted from the synchronous 2D contour maps constructed from the different time domains reveal the kinetics of H/D exchange clearly. The amide protons in the β-turn structures dominate the fast H/D exchange within 6 min after the initiation; followed by the exchanges in the amide protons of another kind of β-turns and the readily accessible part of R-helix structures within 14 min, and in the last part the R-helices are subjected to the H/D exchange.

Hydrogen-Deuterium Exchange of Human Serum Albumin Conclusions The present study has demonstrated that both 2D IR correlation spectroscopy and PCA are powerful to explore the kinetics of the H/D exchanges in HSA and to unravel poorly resolved amide I, II, and II′ regions. Asynchronous spectra are particularly useful to deconvolute amide I, II, and II′ band and to investigate the sequence of the secondary structures exposed to the H/D exchanges. PCA has been employed to separate the different time domains of the H/D exchanges before the calculation of 2D correlation spectra. The combination of PCA and 2D correlation analysis turns out to be very efficient to distinguish the fast kinetics from the slow ones during the H/D exchange process. The loadings plots of PCA have played very important roles in the band assignments and the investigation of the kinetics of the H/D exchange. The present study has revealed the kinetics of the H/D exchanges in HSA as follows: (i) The H/D exchanges proceed in the extended chain and β-turn structures first (a first few minutes after the initiation) and then start occurring in the water accessible parts of the R-helices about 6 min after the start of the H/D exchange. The exchanges in the β-turns continue until 13.4 min after the initiation; (ii) About 25% of the R-helix structures in HSA is still resistant to the H/D exchange even 4 h after the initiation at 25 °C. Acknowledgment. The present study was supported by a Grant-in-Aid to Y. Ozaki (11640516) from the Ministry of Education, Science, and Culture, Japan. References and Notes (1) Clark, R. J. H., Hester, R. E. Eds: Biomolecular Spectroscopy; John Wiley & Sons: Chichester, 1993; Vol. 20, Part A, and Vol. 21, Part B. (2) Havel, H. A. Spectroscopic Methods for Determining Protein Structure in Solution; John Wiley & Sons: Chichester, 1995. (3) Jackson, M.; Mantsch, H. H. Crit. ReV. Biochem. Mol. Biol. 1995, 30, 95. (4) Torii, H.; Tasumi, M. In Infrared Spectroscopy of Biomolecules; Mantsch, H. H., Chapman, D., Eds.; John Wiley & Sons: New York, 1996; p 1. (5) Haris, P. I.; Chapman, D. in Infrared Spectroscopy of Biomolecules; Mantsch, H. H., Chapman, D., Eds.; John Wiley & Sons: New York, 1996; p 239. (6) Goormaghtigh, E.; Cabiaux, V.; Ruysschaert, J.-M. In Subcellular Biochemistry; Hilderson, H. J., Ralston, G. B., Eds.; Plenum Press: New York, 1994; Vol. 23, p 629. (7) Dong, A.; Matsuura, J.; Allison, S. D.; Chrisman, E.; Manning, M. C.; Carpenter, J. F. Biochemistry 1996, 35, 1450. (8) Dong, A.; Hyslop, R. M.; Pringle, D. L. Arch. Biochem. Biophys. 1996, 333, 275. (9) Shaw, R. A.; Buchko, G. W.; Wang, G.; Rozek, A.; Treleaven, W. D.; Mantsch, H. H.; Cushley, R. J. Biochemistry 1997, 36, 14531.

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