Experimental Study on Conformational Changes of Lysozyme in

Nov 9, 2009 - Possible effects of pulsed electric field on proteins have been predicted by ... By use of nonequilibrium molecular dynamics simulations...
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J. Phys. Chem. B 2010, 114, 503–510

503

Experimental Study on Conformational Changes of Lysozyme in Solution Induced by Pulsed Electric Field and Thermal Stresses Wei Zhao and Ruijin Yang* State Key Laboratory of Food Science & Technology and School of Food Science and Technology, Jiangnan UniVersity, No. 1800 Lihu Road, Wuxi 214122, China ReceiVed: August 23, 2009; ReVised Manuscript ReceiVed: October 7, 2009

The responses of proteins to different forms of stress are not well understood at the present time. The “nonthermal” effect of electric field on proteins has recently become an area of great theoretical and practical interest. Possible effects of pulsed electric field on proteins have been predicted by theoretical simulations. However, there is still a lack of experimental reports. In this work, experimental studies were carried out to investigate and compare the effects of 3.5 × 106 V/m pulsed electric field and thermal stresses on the conformational changes of lysozyme in solution. The changes in secondary structure and conformation of disulfide linkage (S-S) of lysozyme were investigated experimentally by circular dichroism analysis and micro-Raman spectra. The motions of tryptophan and tyrosine side chains in lysozyme were also evaluated. From the experimental data, different actions of electric field and thermal stresses on the protein were discovered. 1. Introduction The response of proteins to different forms of stress such as thermal, chemical, pressure, and electric field has always been a topic of major interest, especially the nonthermal effects of electric field on proteins have recently become an area of theoretical and practical interest.1-10 The conformational changes of proteins induced by electric field stress have been investigated by using a series of methods including theoretical simulations1-6 and experimental determinations.7-10 Moreover, pulsed electric field has also been utilized in medicine and molecular biology such as electroporation11 and electrofusion.12 In the food industry, high-intensity pulsed electric field13-15 has been exploited as an alternative to traditional thermal sterilization to inactivate micro-organisms and enzymes. Several researchers also extracted recombinant enzymes16 or DNA17 from microorganisms or tissue with pulsed electric field. In these fields, the effect of pulsed electric field on activity and structure of proteins is also a hot point for research. Currently, many researchers have studied the possible effects of pulsed electric field on proteins by theoretical simulations. Toschi et al.1 employed molecular dynamics simulations to investigate the interaction of β-amyloid peptides with externally applied electric fields. The results suggest that the applied electric field favors the switch of Aβ-peptides from helical to β-sheet conformation, and switching off the field does not restore the original conformation. Budi et al.2-4 performed molecular dynamics simulations on insulin chain-B under the influence of both static and oscillating electric fields, ranging from 107 to 109 V/m. Their studies discovered that both variants had an effect on the normal behavior of the protein; however, the oscillating field was shown to cause complete loss of secondary structure at a lower field intensity as compared to the static electric field, indicating that the rapid change in electric field direction does more damage to the secondary structure than the application of a fixed electric field. English et al.5,6 have * To whom correspondence should be addressed. Phone/Fax: 86 510 85919150. E-mail: [email protected].

performed nonequilibrium molecular dynamics simulations of hen egg white lysozyme at 300 K and 1 bar in the presence of external electric field with varying intensities. Significant nonthermal effects were noted, such as marked changes in the protein’s secondary structure related to protein denaturation. Theoretical modeling is a useful complement to experimental studies in providing insights into the effects of electric field on proteins. However, compared with the extensive studies with theoretical simulations, there are limited experimental reports. Experimental results showed that exposure to electromagnetic radiation could alter protein conformation without bulk heating.7,8 Liu et al.9 used spectrofluorimetry to study the conformational changes of bovine serum albumin exposed to a low frequency electric field. They observed oscillatory fluorescence variations synchronized with the electric field bursts. Zhao et al.10,14 investigated the conformational changes of lysozyme induced by electric field with circular dichroism (CD) and fluorescence spectroscopy. For further insight into the protein response mechanisms to electric field stress, more experimental data on the conformational changes of protein under electric field stress are necessary. Electric field and thermal stresses are different physical stimulations. However, one of the possible explanations for the nonthermal effect of electric field on proteins is that a rapid temperature rise returns to the baseline temperature faster than can be detected by normal thermometry. The temperature rise can alter protein conformation, which in turn can change a protein’s activity, causing detrimental effects.18 It is necessary to be clear the response of protein to both forms of stresses, and it is also useful to examine and compare protein behavior under electric field alone and under thermal stress. Legge et al.19 and Budi et al.3,4 used molecular dynamics to predict the effects of thermal and electric field stresses on the peptide. They found both oscillating electric field and thermal stresses could interfere the normal behavior of a protein, characterized by increasing the mobility and flexibility. By use of nonequilibrium molecular dynamics simulations, English et al.6 discovered 0.25-0.5 V/Å electric fields could induce initial denaturation

10.1021/jp9081189  2010 American Chemical Society Published on Web 11/09/2009

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Figure 1. A schematic diagram of 3-dimensional molecular structure of hen egg white lysozyme in solution (PDB code: 1GXX). The bonds in yellow represent disulfide bonds.

of hen egg white lysozyme to a comparable extent to thermal denaturation in the 400 to 500 K range. In this work, lysozyme was selected as a model to investigate and compare protein behavior under electric field alone and under thermal stress. Lysozyme is one of the most commonly used model systems to study the behavior of proteins. It is a relatively small globular, monomeric protein (Figure 1), containing structure elements (three stretches of R-helix, an antiparallel pleated sheet, and a sequence folded in irregular way) commonly found in proteins. The lysozyme structure is clearly characterized as a compactly folded molecule, the rigidity of which is stabilized by four disulfide bonds (6Cys-127Cys, 30Cys-115Cys, 64Cys-80Cys, and 76Cys-94Cys). Moreover, there are three tyrosine residues and six tryptophan residues in lysozyme, which are associated with the active site and the binding of substrate to lysozyme.20-22 CD is a relatively powerful technique to investigate the secondary structural change of proteins. Raman spectroscopy can complement the standard methods such as mass spectrometry, X-ray crystallography, and NMR spectroscopy to analyze molecular structures. It could give detailed structural information that is not readily available from other techniques. In this work lysozyme in solution was subjected to 3.5 × 106 V/m electric field alone and thermal stress. The conformational changes were studied experimentally with CD and micro-Raman spectra. The aim of the present work was to (i) obtain more detailed information about conformational changes of lysozyme induced by electric field and thermal stresses and (ii) compare the protein’s response to electric field and thermal stresses when the residual activity of lysozyme decreased to a comparable extent induced by the two external physical stresses. The present study confirms and extends the theoretical studies on effects of electric field on proteins using experimental data and further discovers the different behavior of protein under electric field and thermal stresses. 2. Experimental Section 2.1. Chemicals and Materials. Hen egg-white lysozyme was purchased from Amresco Inc. (Solon, OH, USA). Micrococcus lysodeikticus powder (M-3770) was purchased from Sigma Co. (St. Louis, MO). All other chemicals used were of reagent grade. 2.2. Preparation of Lysozyme Solution. Lysozyme powder was dissolved in sodium phosphate buffer (10 mM, pH 6.2) with an electrical conductivity of 0.06 S/m at 25 °C. The concentration of lysozyme solution was 3%.

Zhao and Yang 2.3. Microsecond Pulsed Electric Field Device. A bench scale continuous pulsed electric field system (OSU-4 L, The Ohio State University, OH, USA) with square-wave pulses was used in this work. The pulsed electric field apparatus has been described in our previous study.10 Lysozyme solution flowed through the system to expose to microsecond pulsed electric field with alternating positive and negative pulses when bipolar pulses were applied. A cooling coil with a 2.3-mm inner diameter was connected to each pair of chambers and submerged in a water bath (model 1016, Fisher Scientific Inc., Pittsburgh, PA) to regulate the temperature of lysozyme solution in pulsed electric field system. Type K thermocouples (Fisher Scientific, Pittsburgh, PA) were attached to the surface of the stainless steel coils near the inlet and outlet of each pair of pulsed electric field chambers. The highest temperature in this system was lower than 20 °C. 2.4. Thermal Treatment. For thermal treatment, 18 mL of sodium phosphate buffer was sealed with a butyl septa seal and an aluminum cap into a 25-mL supelco serum type reaction vial and pre-equilibrated in an agitated water bath set at the temperature of 100 °C. Once the phosphate buffer, monitored with a 0.5-mm diameter copper-constantan thermocouple probe (OMEGA Engineering Inc., Stanford, CT, USA), reached the required temperature (100 °C), 2 mL of lysozyme solution (30%) was injected into the vial to reach the concentration (3%). Although this briefly lowered reaction temperature about 4 °C, nominal temperature was re-established within 30 s. A volume of 3 mL was removed immediately from the reaction solution to a test tube at appropriate time intervals and cooled in an ice-water bath until tested for analysis. 2.5. Lysozyme Activity Assay. Lysozyme activity was determined by the turbidimetric assay method as described in our previous study,10 measuring the decrease in absorbance at 450 nm of a M. lysodeikticus suspension vs time with a UV-vis spectrophotometer (UV1201, Beijing Ruili Instrument Co., Beijing, China). A fresh suspension of M. lysodeikticus (18 mg solid in 100 mL of phosphate buffer) was used as substrate. For each sample, 2.3 mL of substrate was placed in a cuvette held at 25 °C. At time 0, 0.3 mL of lysozyme sample, adequately diluted according to its expected activity, was added to give a total reaction volume of 2.6 mL and shaken quickly. Absorbance measurements were made in 0.5-s intervals, the decrease of absorbance vs time were plotted and the activity of each sample was calculated (∆Abs450/min). One enzyme unit is equal to a decrease in turbidity of 0.001/min at 450 nm, the specific activity of per milliliter of lysozyme sample was calculated as follows

Units(U) )

∆Abs450nm/min × 1000 0.3

The relative residual activity (RRA) of lysozyme was defined as a percentage of activity of the lysozyme induced by pulsed electric field or thermal stress relative to that of the control. The control was kept in a 0 °C ice-water bath. Prior to activity assay, all samples were kept in a 0 °C ice-water bath. 2.6. CD Analysis. CD analysis was carried out with a CD spectropolarimeter (Jasco J-715, Jasco Corp., Tokyo, Japan) in the far-UV regions at 25 °C. Quartz CD cuvettes (Hellma, Muellheim, Baden, Germany) with 1-mm path lengths were used. The final lysozyme concentration was adujsted to 2.5 µmol/L with phosphate buffer. Five scans were averaged to obtain one spectrum. The CD data were expressed in terms of molar ellipticity, (θ), in degree cm2/dmol. Estimation of secondary structure was performed using the CDPro suite of

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programs.23 The calculations were made using the three programs included in this software: SELCON3, CONTINLL, and CDSSTR. A basis set containing spectra of 43 proteins was used as data for fitting the experimental spectrum. CDSSTR yielded the best fit values (NRMSD < 0.1 in all cases). 2.7. Micro-Raman Spectra. Micro-Raman spectra were recorded in the range of 300-1800 cm-1 on a LabRam-1B (Dilor, France) instrument coupled with an OLYMPUS BH2 optical microscope. An exciting wavelength of 632.8 nm was provided by a 6.4-mW He-Ne laser source. The Raman spectra were collected and recorded using a Peltier cooled chargecoupled device (CCD) detector with an exposure time of about 100 s. 3. Results and Discussion 3.1. Effects of Pulsed Electric Field and Thermal Stresses on the Activity of Lysozyme. The nonthermal effect of electric field can alter protein conformation, which in turn can change a protein’s activity.1-8 In this work, the activity of lysozyme decreased under both electric field and thermal stresses, which followed a first order model. Specifically, the RRA values of lysozyme as a function of exposure time (t) of 3.5 × 106 V/m electric field were greatly fit to the first-order inactivation model, ln(RRA) ) -0.0004t (µs) + 0.0338 with a correlation coefficient (R2) of 0.9897. Similarly, the first-order inactivation model, ln(RRA) ) -0.0697t (min) + 0.1273 (R2 ) 0.9919) was suitable for describing the inactivation of lysozyme induced by thermal stress. To investigate and compare the conformational changes of lysozyme induced by electric field and thermal stresses, lysozyme samples with various RRA values induced by 3.5 × 106 V/m electric field and thermal stress were prepared. According to the inactivation model, lysozyme samples with RRA values of 92.0, 80.0, 71.0, and 62.0% induced by exposure of 3.5 × 106 V/m electric field for 300, 600, 900, and 1200 µs, respectively, were selected. In the same way, the corresponding lysozyme samples with RRA values of 90.0, 78.0, 71.0, and 63.0% induced by thermal treatment of 2, 4, 6, and 8 min at 100 °C, respectively, also served in the following study. 3.2. Effects of Pulsed Electric Field and Thermal Stresses on the Secondary Structure of Lysozyme. The secondary structure of proteins refers to ordered local structural features within a protein. The far-UV CD spectrum is directly related to the protein secondary structure, due to symmetrical packing of intrinsically achiral (planar) peptide groups.24 Figure 2 illustrates the CD spectra of lysozyme with various RRA values induced by 3.5 × 106 V/m electric field and thermal stresses. The spectra of control display negative CD bands in a wavelength range shorter than 240 nm, which are characterized mainly by two negative bands at 208 and 222 nm. Figure 2a shows that the intensity of the negative peak of 208 nm decreased with the RRA values decrease from 100% (control) to 62.0% induced by electric field. Moreover, the negative peak of 222 nm disappeared with the emergence of a weak positive band between 220 and 230 nm when the RRA decreased to 62.0%. These results indicate the loss of ordered structure (such as R-helix) and an increase of the disorder structure of lysozyme were caused by 3.5 × 106 V/m electric field stress,24 which is in agreement with the result of theoretical modeling5,6 of lysozyme under electric field stress. English et al.5,6 found marked changes in the lysozyme’s secondary structure using nonequilibrium molecular dynamics simulations. They theoretically explained that this occurred primarily as a consequence of alignment of the protein’s total dipole moment with the

Figure 2. CD spectra of lysozyme with various RRA values induced by (a) a 3.5 × 106 V/m electric field and (b) thermal stresses.

external electric field. Figure 2b illustrates the CD spectra of lysozyme with various RRA values from 100% (control) to 63.0% induced by thermal stress. Compared with the effects of electric field stress (Figure 2a), substantial differences in behavior were discovered for lysozyme with RRA values of 90.0% and 78.0% induced by thermal stress. Lysozyme with initial inactivation (such as RRA ) 90. and 78.0%) induced by thermal stress had the same backbone secondary structure as the native protein (Figure 2b). Such conformational state might be viewed as molten globule-like state.25 A molten globule is an intermediate protein structure between native and denatured protein forms, but is distinguished from native proteins by a nonrigid side-chain arrangement and nonfixed tertiary structures.26 When the RRA values decreased to 71 and 63% (Figure 2b), loss of ordered structure (such as R-helix) and an increase of the disorder structure of lysozyme were also caused by thermal stress. The secondary structure of protein can be classified into several categories: helices, sheets, turns, and random coil. Lysozyme, a small monomeric globular protein, has a structure that is compact with several helices surrounding a small β sheet region. It belongs to R and β proteins. The active site is formed at the interface between R and β domains.20 The secondary structure contents of lysozyme as determined from the far-UV CD spectra were calculated. Figure 3 shows the changes of R-helical, β-sheet, and random coil secondary structures of lysozyme with various RRA values induced by 3.5 × 106 V/m electric field and thermal stresses. For the native lysozyme (RRA ) 100%), the R-helical, β-sheet, and random coil contents were approximately 36.0, 13.0, and 29.0%, respectively. As shown in Figure 3a, the R-helical content in lyzozyme decreased linearly from 36.0 to 12.5% with the decrease of RRA from 100 to 62.0%. The β-sheet and random coil contents, on the contrary, increased from 13.0 to 24.0% and from 29.0 to 39.0%,

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Figure 3. The change of R-helical, β-sheet, and random coil secondary structures of lysozyme with various RRA values induced by (a) a 3.5 × 106 V/m electric field and (b) thermal stresses.

respectively. In general, the hydrogen bonding in β-sheet is weaker than that in helix. Therefore, the switch from helical to β-sheet conformation under pulsed electric field stress is associated with the destabilization of the protein secondary structure. The β-sheet is also very important in the processes like aggregation once a hydrophobic contact has been made. Toschi et al.1 found that the presence of an electric field, even for relatively short time, triggered the conversion of Aβ-(1-40) helical to the β-sheet structure, which was ultimately related to the β-amyloid aggregation typical of the Alzheimer’s disease plaques. The decrease of R-helix and increase of random coil demonstrate the loss of ordered structure and an increase of the disorder structure in lysozyme induced by electric field stress. From Figure 3b, it can be seen that there was little change in R-helical, β-sheet, and random coil secondary structures of lysozyme with RRA values more than 80% induced by thermal stress. However, when the RRA values were less than 80%, similar results with Figure 3a were obtained. 3.3. Effects of Pulsed Electric Field and Thermal Stresses on the Conformation of Disulfide Linkage (S-S) of Lysozyme. Micro-Raman spectroscopy of the protein is a reliable tool to monitor the changes in the protein structure and the microenvironment of side chains. It has been used to investigate the structure of proteins.22,27 The micro-Raman spectra of lysozyme with various RRA values induced by 3.5 × 106 V/m electric field and thermal stresses are shown in Figure 4. The assignments of the main bands in the Raman spectra of lysozyme with various RRA values induced by electric field and thermal stresses have been listed in Tables 1 and 2, respectively. Raman spectroscopy is a useful tool for obtaining information on the changes of the sulfur moieties in protein. As shown in

Zhao and Yang

Figure 4. The micro-Raman spectra of lysozyme with various RRA values induced by (A) a 3.5 × 106 V/m electric field and (B) thermal stresses. (a) Control; (b-e) lysozyme with RRA values of 92.0, 80.0, 71.0, and 62.0% induced by 3.5 × 106 V/m electric field stress, respectively; (b′-e′) lysozyme with RRA values of 90.0, 78.0, 71.0, and 63.0% induced by thermal stress, respectively.

Figure 1, the lysozyme structure is stabilized by the four disulfide bonds (6Cys-127Cys, 30Cys-115Cys, 64Cys-80Cys, and 76Cys-94Cys). The Raman spectrum in the region 500-550 cm-1 could be attributed to S-S vibration. Raman bands near 510, 525, and 540 cm-1 due to the S-S stretching mode can be ascribed to gauche-gauche-gauche (g-g-g), gauchegauche-trans (g-g-t), and trans-gauche-trans (t-g-t) conformations of disulfide bonds, respectively.28 Figure 5 shows the 500-550 cm-1 Raman region of lysozyme with various RRA values induced by a 3.5 × 106 V/m electric field and thermal stresses. As shown in Figure 5a, the four S-S bridges in native lysozyme (RRA ) 100%) give rise to two Raman bands near 510 and 530 cm-1, indicating that the four intramolecular S-S bonds in native lysozyme are in g-g-g and g-g-t conformations, which is in agreement with the results of van Wart et al.29 With the decrease of RRA values from 92.0 to 62.0% induced by electric field (Figure 5a), the intensity of the S-S vibration near 530 cm-1 progressively decreased and disappeared with the emergence of band near 540 cm-1, indicating the distortion of g-g-t disulfide bonds and the formation of t-g-t disulfide bonds. When the residual activity decreased to less than 80.0% induced by electric field stress (Figure 5a), the conformations of disulfide bonds in lysozyme have been completely changed to g-g-g and t-g-t conformations from g-g-g and g-g-t conformations. The disulfide bonds are very important and essential for the stability and activity of lysozyme. The transitions of S-S conformation in lysozyme reflect the changes of tertiary structure induced by electric field stress. Figure 5b shows the 500-550-cm-1 Raman region of lysozyme with various RRA values induced by thermal stress. Compared with the results in Figure 5a, different behaviors of disulfide bonds induced by thermal stress were found. No

Conformational Changes of Lysozyme in Solution TABLE 1: Assignment of the Main Bands in the Raman Spectra of Lysozyme with Various RRA Values Induced by 3.5 × 106 V/m Electric Field Stress

J. Phys. Chem. B, Vol. 114, No. 1, 2010 507 TABLE 2: Assignment of the Main Bands in the Raman Spectra of Lysozyme with Various RRA Values Induced by Thermal Stress

Raman wavenumbers (cm-1)

Raman wavenumbers (cm-1)

control

RRA ) 92.0%

RRA ) 80.0%

RRA ) 71.0%

RRA ) 62.0%

510 529

510 521

510

510

510

622 644 671 720 760 830 847 874 931 951 980 1013 1037 1130 1225 1255 1267 1340 1364 1449 1514 1541 1556 1583 1621 1634

622 644 669 720 758 830 846 874 931 949 980 1013 1037 1130 1225 1255 1271 1342 1360 1449 1519 1541 1556 1584 1621

547 622 644 668 724 759 825 842 868 931 953 980 1012 1037 1130 1225 1255 1267 1344 1369 1448 1514 1541 1556 1583 1621 1636 1653 1660 1688

543 620 643 666 724 760 837 853 877 935

545 620 641 667 725 753 825 850 864 935

980 1010 1037 1225 1255 1269 1343 1363 1449 1514 1541 1556 1582 1621

980 1012 1037 1132 1230 1255 1259 1343 1362 1449 1519 1541 1556 1584 1621

1653 1660 1688

1653 1660 1688

1660 1688

1653 1660 1688

assignments

control

RRA ) 90.0%

RRA ) 78.0%

RRA ) 71.0%

RRA ) 63.0%

S-S S-S S-S Phe Tyr, C-S C-S C-S Trp Tyr Tyr Trp C-N C-N, Trp C-N, Tyr Trp, Phe Phe C-N, Trp amide III amide III amide III Trp Trp δCH2, δCH3 His Trp Trp Trp Trp, Tyr, Phe amide I amide I amide I amide I

510 529

510 526

510 535

510

622 644 671 720 760 830 847 874 931 951 980 1013 1037 1130 1225 1255 1267 1340 1364 1449 1514 1541 1556 1583 1621 1634 1660 1688

622 644 669 720 760 830 856 877 931 951 980 1013 1037

620 644 677 720 760 834 845 867 931 951 980 1010 1037 1133 1225 1255 1267 1344 1365 1449 1514 1541 1556 1583 1621 1634 1655 1660 1688

542 621 645 677 720 761 834 850 877 930 951 980 1010 1037 1130 1221 1255 1267 1343 1362 1449 1514 1541 1554 1583 1621

510 525 545 620 649 669 720 760 831 849 878 931 951 980 1011 1037 1130 1225 1255 1264 1345 1370 1449 1514 1541 1554 1583 1621

1650 1660 1688

1652 1660 1688

marked conformational changes were observed in the Raman spectrum of lysozyme with RRA value of 90.0%. However, the S-S vibration was prominent with a marked shifting of the band position from 530 to 540 cm-1 and intensity decreased near 530 cm-1. The data suggest a conformational transition of disulfide bonds from g-g-t to t-g-t conformation was induced by thermal stress. When the RRA value of lysozyme decreased to 63.0% (Figure 5b), the spectrum at 500-550 cm-1 Raman region exhibited a predominant band near 510 cm-1 and two minor bands near 530 and 540 cm-1, indicating the disulfide bonds in lysozyme were predominant in g-g-g conformation with small amount in g-g-t and t-g-t forms. Different from the change in S-S conformation induced by electric field tress, the conformations of disulfide bonds changed to three-form coexisting conformations (g-g-g, g-g-t, and t-g-t conformations) from g-g-g and g-g-t conformations with the decrease of RRA from 100% to 63.0% induced by thermal stress. 3.4. Effects of Pulsed Electric Field and Thermal Stresses on the Motion of Tryptophan (Trp) Side Chains in Lysozyme. Raman spectroscopy has been proved to be a useful technique in revealing conformational changes of proteins, also in the microenvironment of the side chains.22 The aromatic side chains give rise to some interesting features in the spectra, which could reflect changes in the environment of these side chains. There are six Trp residues in lysozyme. Trp-62 and Trp-63 are arranged along one side of the active site, and Trp-108 is in the active cavity. Trp-28 and Trp-111 are in the hydrophobic region, and Trp-123 is located apart from the others. Among the six Trp residues, three (Trp-108, Trp-28, and Trp-111) of them are

1225 1255 1267 1343 1366 1449 1519 1541 1556 1583 1621 1653 1660 1688

assignments S-S S-S S-S Phe Tyr, C-S C-S C-S Trp Tyr Tyr Trp C-N C-N, Trp C-N, Tyr Trp, Phe Phe C-N, Trp amide III amide III amide III Trp Trp δCH2, δCH3 His Trp Trp Trp Trp, Tyr, Phe amide I amide I amide I amide I

buried in the hydrophobic region in native lysozyme.20 The conformational change of lysozyme could be reflected by the motion of side chains such as Trp residues. Some of the previously buried side chains in the interior of the compact globular region of native lysozyme protein might be exposed to the surface of molecular, a more polar environment under some stresses. In a contrary manner, some side chains located in the surface might be surrounded with other chains, forming a more hydrophobic environment. The micro-Raman bands near 1340 and 1360 cm-1 are both assigned to the Trp residues due to the Fermi resonance between one skeletal stretching fundamental and one or two combinations of the indole ring vibrations.27,30,31 The Raman signature from tryptophan residues is a Raman indicator, of which the intensity ratio I1360/I1340 is particularly sensitive to the transition of Trp microenvironments from a hydrophobic one to a hydrophilic one accompanying the inactivation of lysozyme, because hydrophobic interactions between the indole ring of the Trp residue and the surrounding aliphatic groups enhance the peak at 1360 cm-1 and reduce the one near 1340 cm-1.27,30,31 Figure 6 illustrates the1340-1380 cm-1 Raman region of lysozyme with various RRA values induced by 3.5 × 106 V/m electric field and thermal stresses. The ratio (I1360/I1340) for native lysozyme (RRA ) 100%) was calculated as 1.18, which was close to 1.04 calculated by shou et al.,27 indicating that most Trp residues are buried in the hydrophobic region in native lysozyme. However, remarkable intensity changes were observed for lysozyme with various RRA values near 1340 and 1360 cm-1, revealing the change in threedimensional positions and the motion of Trp residues of

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Figure 6. 1340-1380-cm-1 Raman region of lysozyme with various RRA values induced by (a) a 3.5 × 106 V/m electric field and (b) thermal stresses.

Figure 5. 500-550-cm-1 Raman region of lysozyme with various RRA values induced by (a) 3.5 × 106 V/m electric field and (b) thermal stresses.

lysozyme induced by 3.5 × 106 V/m electric field stress. In particular, a strong increase in the intensity of band at 1340 cm-1 and decrease in that of band at 1360 cm-1 made the sharp decline in ratio (I1360/I1340) of Fermi doublet of Trp as a consequence of electric field stress. On the basis of the results and calculation, the ratios (I1360/I1340) for lysozyme with RRA of 92.0, 80.0, 71.0, and 62.0% (Figure 6a) decreased to 0.51, 0.67, 0.78, and 0.54 from 1.18, respectively. From these data, we can see that all the intensity ratios (I1360/I1340) of Fermi doublet of Trp decreased for lysozyme with different RRA values, indicating that a decrease in the hydrophobicity of the Trp environment, presumably because some of the previously buried Trp residues were exposed to a more polar environment. However, the decreases seem to be random and not in a trend like the RRA values. It is probably probably because the motion of Trp residues could just act as one of the indicators of conformational changes of lysozyme as a consequence of electric field stress, whereas the alteration in tertiary structure of protein is actually more complicated. For lysozyme with various RRA values induced by thermal stress (Figure 6b), remarkable changes were also observed for

the doublet bands at 1340 and 1360 cm-1and similar results were obtained. The ratios (I1360/I1340) for lysozyme with RRA of 90.0, 78.0, 71.0, and 63.0% sharply decreased to 0.42, 0.54, 0.60, and 0.68 from 1.18, respectively. This demonstrates a similar trend of conformational changes induced by thermal stress, suggesting an increase in the hydrophilicity of the Trp environment. It is known that Trp-108 is located at the active cavity and Trp-62 is arranged along one side of the active site. When the enzyme-substrate complex is undergoing catalysis, the NH in indole ring of Trp-62 is hydrogen bonded to the oxygen attached to C-6 of tri-N-acetylglucosamine, the substrate of lysozyme.32 Therefore, the change in locations of Trp-62 and Trp-108 could be reflective of conformational change of the active site and inhibition of the binding of substrate to lysozyme. The distortion of g-g-g disulfide bonds could also be associated with the changes observed in the Trp bands. According to the X-ray structural analysis of lysozyme, the Cys64-Cys80 is one of the three disulfide bonds with a g-g-g conformation and a relationship between conformational changes in Cys64-Cys80 and Trp-62 and -63 has been established. Therefore, distortion of this disulfide bridge will certainly change the environment of these neighboring residues, primarily Trp62 and -63.22 3.5. Effects of Pulsed Electric Field and Thermal Stresses on the Motion of Tyrosine (Tyr) Side Chains in Lysozyme. Tyr side chains are frequently hydrogen bonded to other residues in proteins and can be used as probes of local tertiary structure environment. Among many Tyr Raman bands, the doublet bands located near 830 and 850 cm-1 are useful for determining the

Conformational Changes of Lysozyme in Solution microenvironment around Tyr side chains and the state of hydrogen bonding involving the Tyr OH group.33 This Fermi resonance is assigned to vibrations of the para-substituted benzene ring of Tyr residues which are affected by the environment and the involvement of the phenolic hydroxyl group in hydrogen bonding. Basically two major classes of hydrogen bonding patterns affect the doublet intensities. In one, phenolic oxygen acts as a strong proton donor, with a resulting intensity decrease of the ratio, I850/I830 ≈ 0.5. This has been suggested to be the case for Tyr residues buried within the protein. On the other hand, the phenolic oxygen acts as a much weaker proton donor or as an acceptor to an external acidic proton, I850/I830 lies near 1.25, indicating Tyr residues are exposed to the aqueous or polar environment.34,35 If intensity ratio of the doublet bands (I850/I830) ranges from 0.5 to 1.25, the distribution of Tyr residues in “buried” or “exposed” environments could be calculated from the Raman data according the following35,36

0.5Nb + 1.25Ne ) I850/I830 Nb + Ne ) 1 where Nb and Ne are the mole fractions of buried and exposed Tyr residues, respectively. The intensity ratio I850/I830 of native lysozyme is 1.12, suggesting that the Tyr residues are mainly exposed and able to participate in moderate or weak hydrogen bonding. According to the calculation, Ne for native lyzozyme is about 0.8. As is known, there are three Tyr residues in lysozyme, Two (Tyr-20 and Tyr-23) of them in the helical domain and one (Tyr-53) in the beta domain.37 Thus we know from the Raman data that, among the three Tyr residues, only one Tyr residue is buried in the hydrophobic region in native lysozyme. This is in agreement with the results determined by X-ray.37 As shown in Figure 4A and Table 1, the peaks near 830 cm-1 of lysozyme with RRA values of 92.0, 80.0,71.0, and 62.0% induced by electric field stress shifted to 830, 825, 837, and 825 cm-1, respectively, and those near 850 cm-1 shifted to 846, 842, 853, and 850 cm-1, respectively. This indicates the change in three-dimensional positions and the motion of Tyr residues of lysozyme. In particular, the intensity ratio (I850/I830) of the doublet at 850-830 cm-1 decreased to 0.62, 1.01, 0.83, and 0.73 for lysozyme with RRA values of 92.0, 80.0, 71.0, and 62.0% induced by electric field stress, respectively. The decreases imply that more Tyr residues were located in hydrophobic environment and mainly acted as a hydrogen-bond donor. Similar to the intensity ratios (I1360/I1340) of Fermi doublet of Trp residues, the decreases of the intensity ratios (I850/I830) of Tyr doublet also do not display in a trend like the RRA values. When the RRA value of lysozyme decreased to 62.0% induced by electric field stress, the value of Ne decreased to about 0.3, demonstrating that two of the three Tyr residues were buried in the hydrophobic region. For lysozyme with RRA values of 90.0, 78.0, 71.0, and 63.0% induced by thermal stress (Figure 4B and Table 2), the peaks near 830 cm-1 appeared at 830, 834, 834, and 831 cm-1, respectively, and the peaks near 850 cm-1 shifted to 856, 845, 850, and 849 cm-1, respectively. It is interesting to notice the sharply enhancement in the intensity ratio of the Tyr doublet. After thermal treatments, all ratios I850/I830 exceeded 2.0, suggesting that all the three Tyr residues in lysozyme were exposed to the solvent induced by thermal stress. A similar behavior of Tyr residues was observed after heat treatment of lyzozyme in aqueous by Torreggiani et al.38 and Ionov et al.39

J. Phys. Chem. B, Vol. 114, No. 1, 2010 509 Tyr frequently plays a key role in proteins through hydrogen bonding of the hydroxyl group.33 The motions of Tyr side chains in lysozyme during the unfolding of tertiary structure under external stresses are related to the activity change. Since Tyr53 is hydrogen bonded with the amino group of Asp-66 and is adjacent to the catalytic residue Asp-52, the changes in the Tyr environment can be associated both with the modification of the environment of the neighboring Trp residues (Trp-62 and -63) and the influence on the enzymatic active site of lysozyme.40 In the present work, it is worth noting that while the RRA values of lysozyme induced by electric field and thermal stresses were similar, the conformational changes were different, especially more Tyr side chains were buried in the hydrophobic region induced by electric field stress, whereas all the three Tyr residues in lysozyme were exposed to the solvent induced by thermal stress. This indicates the different actions of electric field and thermal stresses on the protein. 4. Conclusions In this work, experimental studies were carried out to investigate and compare the effects of 3.5 × 106 V/m pulsed electric field and thermal stresses on the conformational changes of lysozyme in solution. Experimental data confirmed marked changes in the lysozyme’s secondary structure induced by electric field stress. The conversion from helical to β-sheet and random coil conformations under electric field stress was verified, which was associated with the destabilization of the protein secondary structure. Compared with the effects of electric field stress, substantial differences in behavior of lysozyme secondary structure induced by thermal stress were found. Lysozyme with initial inactivation induced by thermal stress had the same backbone secondary structure as the native protein, which might be viewed as molten globulelike state. Micro-Raman spectroscopy was further employed to study the effects of electric field and thermal stresses on the conformations of disulfide linkage, the motions of Trp and Trp side chains in lysozyme. Our results suggest that the conformations of disulfide bonds in lysozyme had been completed changed to g-g-g and t-g-t conformations from g-g-g and g-g-t, and some of the previously buried Trp residues were exposed to a more polar environment, but more Tyr residues were buried in hydrophobic environment and mainly acted as a hydrogenbond donor induced by 3.5 × 106 V/m pulsed electric field. Different from the action of electric field, the conformations of disulfide bonds changed to three-form coexisting conformations (g-g-g, g-g-t, and t-g-t conformations) from g-g-g and g-g-t conformations, and more Trp residues and all the three Tyr residues in lysozyme were located at the surface of protein molecule induced by thermal stress. These conformational changes presented in this work are associated with the conformational change of the active site, the inhibition of the binding of substrate to protein, and the destabilization of the protein structure induced by electric field and thermal stresses, which ultimately decrease the protein’s activity. The different conformational changes induced by electric field and thermal stresses indicate the different actions of the two external physical stresses on the protein. Acknowledgment. The authors gratefully acknowledge the financial support provided by National 863 Hi-Tech R&D Plan (2007AA100405) and Project 20772049 of the National Natural Science Foundation of PR China National. This study was also supported by 111 project-B07029, Program for Changjiang Scholars and Innovative Research Team in University and the Graduate Student Innovation Project (Jiangsu, China).

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