- Email: [email protected]
Raman study of the cleavage of disulphide bonds in albumin, chymotrypsin, and thrombin
Accepted Manuscript Title: Raman study of the cleavage of disulphide bonds in albumin, chymotrypsin, and thrombin Authors: N.N. Brandt, A.Yu. Chikishev, V.N. Kruzhilin PII: DOI: Reference:
S0924-2031(16)30144-8 http://dx.doi.org/doi:10.1016/j.vibspec.2016.12.005 VIBSPE 2664
To appear in:
VIBSPE
Received date: Revised date: Accepted date:
8-7-2016 20-12-2016 20-12-2016
Please cite this article as: N.N.Brandt, A.Yu.Chikishev, V.N.Kruzhilin, Raman study of the cleavage of disulphide bonds in albumin, chymotrypsin, and thrombin, Vibrational Spectroscopy http://dx.doi.org/10.1016/j.vibspec.2016.12.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Raman study of the cleavage of disulphide bonds in albumin, chymotrypsin, and thrombin
N.N. Brandta, A. Yu. Chikisheva, and V.N. Kruzhilina*
a
Faculty of Physics and International Laser Center, Lomonosov Moscow State University,
Moscow, 119991 Russia *Corresponding author: [email protected], tel. +79168208001
ABSTRACT The structural changes resulting from the reduction of disulphide bonds in three globular proteins (albumin, chymotrypsin and thrombin) in aqueous solutions are investigated using Raman spectroscopy. Changes in SS and SH vibrational bands are used for direct observation of the disulphide bond reduction. The corresponding changes in secondary structure and H-bonding of tyrosines are observed. It is shown that the unfolding pathway after the reduction of disulphide bridges in albumin depends on the reducing agent. The most developed changes are observed for the band assigned to the ggg conformation of the disulphide bridge in two similar serine proteases (thrombin and chymotrypsin) but the secondary structure of thrombin is much more stable against the reduction of the disulphide bonds.
Keywords: Raman spectroscopy; disulphide bridges; protein unfolding; serine proteases; serum albumins;
1. INTRODUCTION Disulphide bonds (bridges) between two cysteins of a polypeptide chain are important for protein stability and activity. CS–SC (), СС–SS (), and SS–CC () dihedral angles are used to characterize conformations of disulphide bridges. Three combinations of dihedral angles , , ≠ 0; 0, , ≠ 0; and , 0, ≠ 0 correspond to gauche-gauche-gauche (ggg), transgauche-gauche (tgg), and trans-gauche-trans (tgt) conformations, respectively [1–3].
1
Raman scattering spectroscopy is known to be a helpful tool in the study of protein structure in aqueous solutions. Raman bands peaking at 490–560 cm-1 are assigned to SS stretching mode of cystine in protein structure. Band positions 500–515, 520–530, and 540–550 cm-1 correspond to the ggg, tgg, and tgt conformations, respectively [1,4,5]. Amide I band (~1650 cm-1) is used to determine secondary structure of proteins [6–8]. Thrombin is a serine protease that plays the key role in blood coagulation, since it converts fibrinogen to fibrin in coagulation cascade. Bovine thrombin is a globular protein, consisting of two polypeptide chains (m.w. 37 kDa). The chains are linked by Cys1–Cys122 disulphide bond. The heavier chain, which is stabilized by Cys42–Cys58, Cys168–Cys182, and Cys191–Cys220 disulphide bridges, contains alpha-helical and beta-sheet fragments [9]. Cleavage of the four disulphide bonds leads to complete denaturation of the protein [10]. The Cys191–Cys220 bond controls the enzymatic activity of thrombin, since it determines the structure of the specificity pocket. The cleavage of the bond leads to a ~100-fold loss of the enzymatic activity [11]. Biochemical methods can hardly be used to comprehensively characterize changes of the remaining disulphide bonds that may indirectly affect the functional activity of thrombin [12]. Note insufficient data on the effect of disulphide bonds on thrombin functioning. Chymotrypsin is a well-studied serine protease (m.w. 25 kDa). Bovine chymotrypsin is a globular protein consisting of three polypeptide chains linked by Cys1–Cys122 and Cys136–Cys201 disulphide bridges. The chains are internally stabilized by Cys42–Cys58, Cys168–Cys182, and Cys191–Cys220 bonds. Beta sheets are dominant elements of chymotrypsin secondary structure [13,14]. The Cys191–Cys220 bond stabilizes the specificity pocket. The mutant enzyme without such a bridge exhibits a decrease in the enzymatic activity by several orders of magnitude [15]. Note similarity of the structural features of chymotrypsin and thrombin. In particular, four of five disulphide bridges in chymotrypsin (Cys1–Cys122, Cys42–Cys58, Cys168–Cys182, and Cys191– Cys220) are fully homologous to the four disulphide bonds of thrombin [16]. Thus, chymotrypsin can be compared with thrombin with respect to the role of disulphide bonds in stabilization of protein structure. Serum albumin is the main protein of blood plasma that provides unspecific bonding and transportation of fatty acids, steroids, and hormones in blood. It is widely employed as a model object in the study of the properties of globular proteins (m.w. 66.5 kDa). The polypeptide chain forms an asymmetric globule, in which α-helix is the dominant structure. The protein structure is stabilized by 17 disulphide bonds, six of which are located in the immediate vicinity of the binding sites [17]. The cleavage of all disulphide bonds in human serum albumin leads to 2
complete disorganization of tertiary structure [18]. Raman spectroscopy has been used to study conformational changes of disulphide bridges in BSA [19] and modification of α-helices due to cleavage of disulphide bonds [7]. The purposes of this work are the comparative study of the reduction of disulfide bonds in albumin, chymotrypsin, and thrombin in aqueous solutions using Raman spectroscopy, the analysis of the effect of the cleavage of disulphide bonds on the secondary structure and the Hbonding of tyrosine residues, and the study of the effect of two reducing agents on albumin. 2. MATERIALS AND METHODS 2.1. Samples In the experiments, we use lyophilized bovine thrombin (Sigma-Aldrich, CN T4648), lyophilized chymotrypsin (Samson-Med), and lyophilized BSA (MP Biomedicals, CN 160069). The proteins are dissolved in Milli-q water at a concentration of 100 mg/mL (1.5 mM for albumin, 3.8 mM for chymotrypsin, and 3.1 mM for thrombin) at pH 7.4. Native structure of thrombin, chymotrypsin, and albumin is not violated at pH 5–10 [20], 7–9 [21], and 5.0–8.0 [22], respectively. Dithiothreitol (DTT) at a concentration of 35 mM and tris(2carboxyethyl)phosphine (TCEP) at a concentration of 35 mM serve as reducing agents: Scheme 1. The DTT interaction with protein yields a reaction product (4,5-dihydroxy-1,2-dithiane (oxidized DTT)) that has an internal disulphide bond. For this reason, we do not use DTT in the experiments with thrombin and chymotrypsin. DTT is employed in the experiments with albumin for comparison with the available Raman data. Aqueous solution of NaOH is used for pH adjustment in the experiments with TCEP. The measurements are performed at room temperature immediately after adding reducing agents, and the evolution of spectra in time is analyzed. The experiments show the absence of variations in the spectra of pure proteins in time under laser irradiation in the course of measurements. 2.2. Raman measurements In the experiments, we use a high-sensitivity Raman spectrometer equipped with a Coherent Innova 90 cw argon-ion laser (excitation wavelength, 514 nm; power on the sample, up to 100 mW; spectral resolution, 2 cm-1; and measurement interval, 200–2000 cm-1 [23] ) and a ThermoScientific MicroRaman DXR Raman microscope (excitation wavelengths, 532 and 780 nm; excitation powers, 10 and 24 mW, respectively; spectral resolution, 2 cm-1; and 3
measurement interval, 40–3500 cm-1). The Raman microscope, which has a lower sensitivity, is used for simultaneous measurements of the Raman bands assigned to SS and SH stretching modes. To assess possible effects of laser radiation on the samples under study, we preliminary performed 4-hour measurements of the spectra of pure protein solutions at the highest laser power (100 mW). The absence of spectral changes in such measurements allows the spectroscopic analysis of changes caused by the cleavage of disulphide bonds. 2.3. Data processing A multistage procedure is used for data processing. First, the spectra are smoothed using the Savitsky-Golay filter [24] and the second derivatives of the spectral curves are calculated to eliminate the background contribution in the subsequent normalization procedure. Original spectra are normalized by the intensity of the band peaking at 1004–1006 cm-1 in the secondderivative spectrum. (The Raman band at 1004–1006 cm-1 is assigned to the breathing mode of the phenylalanine ring.) For the analysis of the amide I band, we perform normalization by the integral intensity of this band. The backgrounds are subtracted using a procedure of [25], which employs the iterative subtraction of the polynomial background. We use the fifth-power polynomials and separately perform background subtraction in intervals of 350–1030, 980–1800, and 2000–2800 cm-1. 3. RESULTS AND DISCUSSION First, we study Raman spectra of albumin in the presence of DTT that can be compared with the results of [7]. Figure 1. Figure 2. Figure 1a compares the spectra of albumin and albumin with DTT. It is seen that the quantitative analysis of the spectral changes is impeded due to significant contribution of the signal of oxidized DTT. Nevertheless, we observe a gradual decrease in the intensity of the bands of the disulphide bridges of the protein, which directly proves the reduction of disulphide bonds (Figure 1b). Note that the reduction takes place for all basic conformations of the bridges. A detailed analysis of the spectral changes is possible with the aid of spectral deconvolution [7]. The cleavage of disulphide bonds is accompanied by minor variations in the relative intensities of the tyrosine doublet (I850/I830) and a shift of the high-frequency component of the doublet (Fig. 2a). Such spectral changes indicate modification of the H-bonds of the tyrosine residues [8,26,27] and, hence, conformational changes of the protein molecule. 4
Note variations in the intensities of the bands assigned to CC skeletal vibrations of the polypeptide change (Fig. 2b). A decrease in the intensity of the band at 940 cm-1 upon thermal denaturation has been observed in [28] and interpreted as a decrease in the amount of -helices. The minor changes in a spectral interval of 990–1000 cm-1 are presumably due to the contribution of oxidized DTT signal. The spectral changes in the range of the amide I band (Fig. 2c) correspond to degradation of the secondary structure, since the high-frequency shift of the band and a decrease in the intensity can be interpreted as a decrease in the amount of -helices and an increase in the amount of randomcoil fragments. Similar changes of the amide I band have been observed in [7]. Figure 3. Figure 3 compares the spectra of albumin and albumin with TCEP. A decrease in the intensity of Raman band at 500 cm-1 proves the dominant cleavage of disulphide bonds in the ggg conformation (Fig. 3a). A minor increase in the signal intensity at frequencies corresponding to the tgg and tgt conformations may indicate that the cleavage of the SS bonds in the ggg conformation is accompanied by partial transformation into less stable tgg and tgt conformations. As in the case of interaction with DTT, cleavage and transformation of disulfide bonds leads to modification of the H-bonds of tyrosine residues , which follows from intensity variations of the tyrosine doublet and a shift of the band peaking at 850 cm-1 (Fig. 3b). We also observe a shift of the amide I band and a decrease in the intensity at 1650 cm-1 (Fig. 3c). Changes of the band assigned to the CC skeletal vibrations can hardly be analyzed, since the corresponding Raman band is overlapped with the TCEP band. After 3 h of interaction with TCEP, the solution becomes turbid presumably due to aggregation of albumin and a significant increase in the background signal impedes Raman measurements. Figure 4. TCEP causes reduction of disulfide bonds of chymotrypsin, and (as in the case of albumin) the most developed spectral changes are observed at frequencies corresponding to the ggg conformation (Fig. 4a). The cleavage of disulfide bonds is additionally proven by an increase in the intensity of the Raman signal corresponding to the SH stretching mode (about 2580 cm-1, Fig. 4c) [29]. Raman band at 2450 cm-1 in the samples with TCEP may correspond to the PH stretching mode of TCEP molecule [30].
5
Changes of the secondary structure resulting from the cleavage of disulfide bonds lead to spectral changes in the range of the amide I band (Fig. 4b). We observe a decrease in the intensity at 1670 cm-1 and an increase in the intensity of the high-frequency components that can be interpreted as partial transformation of sheets into random coils. Beta structure of chymotrypsin is stabilized by three disulphide bonds in the ggg (Cys42–Cys58 and Cys136–Cys201) and tgt (Cys168–Cys182) conformations [13,14]). Apparently, the observed spectral changes are due to cleavage of such bonds. Figure 5. Fig. 5a shows a significant decrease in the signal intensity in the spectral interval of the SS bonds and a corresponding increase in the intensity of the band assigned to the SH stretching mode. The strongest spectral changes are observed in the interval of the disulphide bridges in the ggg conformation as in experiments with chymotrypsin. Again, a minor increase in the signal intensity at frequencies corresponding to the tgg conformation may indicate that the cleavage of the SS bonds in the ggg conformation is accompanied by partial transformation into less stable tgg conformation. Consistent changes are observed in the SH vibration region (Fig. 5c). In comparison with chymotrypsin, thrombin exhibits smaller changes of the amide I band that can be interpreted as partial transformation of helices into sheets (Fig. 5b). Based on the Raman data, we may assume that the structure of thrombin is more stable against cleavage of disulfide bonds. The X-ray data show that two of four disulfide bridges in thrombin have ggg conformation (Cys168–Cys182 and Cys122–Cys1 (interchain)). The results of [10] show that the Cys168–Cys182 bond is the first to be reduced due to interaction with DTT. The cleavage of this bond may lead to changes of the α-helical and β-sheet fragments containing Cys168 and Cys182, respectively. Such changes are in agreement with the results of Fig. 5b. Minor changes in the relative intensities of the tyrosine doublet in the experiments with thrombin and chymotrypsin indicate insignificant changes of the corresponding H-bonds due to the cleavage of disulphide bonds in the two proteins. 4. CONCLUSIONS The cleavage of disulfide bonds in proteins in aqueous solutions can be monitored using variations in the intensities of the Raman bands assigned to the SS and SH stretching modes. The experiments with albumin (evolution of the bands assigned to disulphide bridges and tyrosine doublet) show that the application of different reducing agents leads to different unfolding 6
pathways. The known decrease in the catalytic activity of chymotrypsin related to the cleavage of disulphide bonds may result from both direct changes of the active site and noticeable modification of the secondary structure. Based on the results for the three proteins, we may conclude that disulfide bridges control conformational stability of each protein molecule as a whole and the cleavage of disulfide bonds may lead to less ordered structures with greater contents of random coils. The most developed changes in the spectra of two serine proteases (chymotrypsin and thrombin) in the presence of TCEP are observed at frequencies corresponding to the ggg conformation. Note that the cleavage of disulphide bridges in the serine proteases causes substantially different changes of the secondary structure.
ACKNOWLEDGMENTS We are grateful to A.V. Golovin, Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University for helpful discussions. This work was supported in part by the Russian Foundation for Basic Research (project no. 15-02-06512) and Program for Development of Moscow State University.
REFERENCES [1] H. Sugeta, A. Go, T. Miyazawa, Vibrational Spectra and Molecular Conformations of Dialkyl Disulfides, Bull. Chem. Soc. Jpn. 46 (1973) 3407–3411. doi:10.1246/bcsj.46.3407. [2] W. Zhao, S. Krimm, Vibrational studies of the disulfide group in proteins: Part II. Ab initio force fields and normal mode frequencies of dimethyl, methylethyl, and diethyl disulfides, J. Mol. Struct. 224 (1990) 7–20. doi:10.1016/0022-2860(90)87003-G. [3] C.H. Görbitz, Conformational properties of disulphide bridges. 2. Rotational potentials of diethyl disulphide, J. Phys. Org. Chem. 7 (1994) 259–267. [4] H.E. Van Wart, H.A. Scheraga, Agreement with the disulfide stretching frequencyconformation correlation of Sugeta, Go, and Miyazawa, Proc. Natl. Acad. Sci. 83 (1986) 3064–3067. [5] W. Qian, S. Krimm, Vibrational studies of the disulfide group in proteins. Part V. Correlation of SS stretch frequencies with the CCSS dihedral angle in known protein disulfide bridges, Biopolymers. 32 (1992) 321–326. [6] J.T. Pelton, L.R. McLean, Spectroscopic Methods for Analysis of Protein Secondary Structure, Anal. Biochem. 277 (2000) 167–176. doi:10.1006/abio.1999.4320.
7
[7] C. David, S. Foley, C. Mavon, M. Enescu, Reductive unfolding of serum albumins uncovered by Raman spectroscopy, Biopolymers. 89 (2008) 623–634. doi:10.1002/bip.20972. [8] D. Nemecek, J. Stepanek, G.J. Thomas, Raman Spectroscopy of Proteins and Nucleoproteins, in: J.E. Coligan, B.M. Dunn, D.W. Speicher, P.T. Wingfield (Eds.), Curr. Protoc. Protein Sci., John Wiley & Sons, Inc., Hoboken, NJ, USA, 2013. [9] B.J. Desai, R.S. Boothello, A.Y. Mehta, J.N. Scarsdale, H.T. Wright, U.R. Desai, Interaction of thrombin with sucrose octasulfate, Biochemistry (Mosc.). 50 (2011) 6973– 6982. doi:10.1021/bi2004526. [10] R. Rajesh Singh, J.-Y. Chang, Structural stability of human α-thrombin studied by disulfide reduction and scrambling, Biochim. Biophys. Acta BBA - Proteins Proteomics. 1651 (2003) 85–92. doi:10.1016/S1570-9639(03)00238-3. [11] L.A. Bush-Pelc, F. Marino, Z. Chen, A.O. Pineda, F.S. Mathews, E. Di Cera, Important Role of the Cys-191 Cys-220 Disulfide Bond in Thrombin Function and Allostery, J. Biol. Chem. 282 (2007) 27165–27170. doi:10.1074/jbc.M703202200. [12] A.A. Serejskaya, S.V. Zubak, T.W. Osadchuk, S.B. Serebryani, Enzymic activity of thrombin with partially reduced disulfide bonds, Thromb. Res. 32 (1983) 147–154. [13] K. Brady, A.Z. Wei, D. Ringe, R.H. Abeles, Structure of chymotrypsin-trifluoromethyl ketone inhibitor complexes: comparison of slowly and rapidly equilibrating inhibitors, Biochemistry (Mosc.). 29 (1990) 7600–7607. [14] A. Razeto, B. Galunsky, V. Kasche, K.S. Wilson, V.S. Lamzin, High resolution structure of native bovine alpha-chymotrypsin, Be Publ. (n.d.). doi:10.2210/pdb1yph/pdb. [15] Varallyay E., Lengyel Z., Graf L., Szilagyi L., The Role of Disulfide Bond C191-C220 in Trypsin and Chymotrypsin, BBRC. (1997) 592–596. [16] R.W. Colman, Hemostasis and Thrombosis: Basic Principles and Clinical Practice, Lippincott Williams & Wilkins, 2006. [17] K.A. Majorek, P.J. Porebski, A. Dayal, M.D. Zimmerman, K. Jablonska, A.J. Stewart, M. Chruszcz, W. Minor, Structural and immunologic characterization of bovine, horse, and rabbit serum albumins, Mol. Immunol. 52 (2012) 174–182. doi:10.1016/j.molimm.2012.05.011. [18] G. Markus, F. Karush, The Disulfide Bonds of Human Serum Albumin and Bovine γGlobulin1, J. Am. Chem. Soc. 79 (1957) 134–139. [19] K. Nakamura, S. Era, Y. Ozaki, M. Sogami, T. Hayashi, M. Murakami, Conformational changes in seventeen cystine disulfide bridges of bovine serum albumin proved by Raman spectroscopy, FEBs Lett. 417 (1997) 375–378. [20] R. Machovich, Thrombin The, CRC-Press, 1984. [21] H.U. Bergmeyer, K. Gawehn, Methods of enzymatic analysis, Verlag Chemie, 1974. [22] T. Peters Jr, All about albumin: biochemistry, genetics, and medical applications, Academic press, 1995. [23] N.N. Brandt, A.Y. Chikishev, Laser Raman spectrometer for the studies of biomolecules with monitoring temperature of the samples, Laser Phys. 12 (2002) 647–652. [24] A. Savitzky, M.J.E. Golay, Smoothing and Differentiation of Data by Simplified Least Squares Procedures., Anal. Chem. 36 (1964) 1627–1639. doi:10.1021/ac60214a047. 8
[25] C.A. Lieber, A. Mahadevan-Jansen, Automated method for subtraction of fluorescence from biological Raman spectra, Appl. Spectrosc. 57 (2003) 1363–1367. [26] M.N. Siamwiza, R.C. Lord, M.C. Chen, T. Takamatsu, I. Harada, H. Matsuura, T. Shimanouchi, Interpretation of the doublet at 850 and 830 cm-1 in the Raman spectra of tyrosyl residues in proteins and certain model compounds, Biochemistry (Mosc.). 14 (1975) 4870–4876. [27] J.L. McHale, Fermi resonance of tyrosine and related compounds. Analysis of the Raman doublet, J. Raman Spectrosc. 13 (1982) 21–24. [28] V.J.C. Lin, J.L. Koenig, Raman studies of bovine serum albumin, Biopolymers. 15 (1976) 203–218. doi:10.1002/bip.1976.360150114. [29] H. Li, G.J. Thomas, Studies of virus structure by Raman spectroscopy. Cysteine conformation and sulfhydryl interactions in proteins and viruses. 1. Correlation of the Raman sulfur-hydrogen band with hydrogen bonding and intramolecular geometry in model compounds, J. Am. Chem. Soc. 113 (1991) 456–462. doi:10.1021/ja00002a012. [30] N.B. Colthup, L.H. Daly, S.E. Wiberley, Introduction to Infrared and Raman Spectroscopy, Elsevier, 1990.
9
FIGURE AND SCHEME CAPTIONS Figure 1. (a) Raman spectra (ex = 514.5 nm) of (solid line) albumin in aqueous solution and (dashed line) oxidized DTT and (b) spectra of albumin solutions with DTT measured over three subsequent time intervals of 80 min.
10
Figure 2. (Upper curves) Raman spectra (ex = 514.5 nm) of (black line) albumin aqueous solution and albumin solutions with DTT measured over time intervals of (dark gray line) 0–80, (grey line) 80–160, and (light grey line) 160–240 min. and (lower curves) scaled-up differences of the above spectra and the spectrum of albumin aqueous solution.
11
Figure 3. (Upper curves) Raman spectra (ex = 514.5 nm) of (black line) albumin aqueous solution and albumin solutions with TCEP measured over time intervals of (dark gray line) 0–40, (grey line) 40–80, (light grey line) 80–120, and (dashed line) 120–160 min. and (lower curves) scaled-up differences of the above spectra and the spectrum of albumin aqueous solution.
12
Figure 4. (Upper curves) Raman spectra (ex = 532 nm) of (black line) chymotrypsin aqueous solution and albumin solutions with TCEP measured over time intervals of (dark gray line) 40– 80, (grey line) 120–160, and (light grey line) 200–240 min. and (lower curves) scaled-up differences of the above spectra and the spectrum of albumin aqueous solution.
13
Figure 5. (Upper curves) Raman spectra (ex = 780 nm) of (black line) thrombin aqueous solution and albumin solutions with TCEP measured over time intervals of (grey line) 0–120, and (dashed line) 120–240 min. and (lower curves) scaled-up differences of the above spectra and the spectrum of albumin aqueous solution.
14
Scheme 1. Reduction of protein disulphide bonds with DTT and TCEP.
15
S0924-2031(16)30144-8 http://dx.doi.org/doi:10.1016/j.vibspec.2016.12.005 VIBSPE 2664
To appear in:
VIBSPE
Received date: Revised date: Accepted date:
8-7-2016 20-12-2016 20-12-2016
Please cite this article as: N.N.Brandt, A.Yu.Chikishev, V.N.Kruzhilin, Raman study of the cleavage of disulphide bonds in albumin, chymotrypsin, and thrombin, Vibrational Spectroscopy http://dx.doi.org/10.1016/j.vibspec.2016.12.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Raman study of the cleavage of disulphide bonds in albumin, chymotrypsin, and thrombin
N.N. Brandta, A. Yu. Chikisheva, and V.N. Kruzhilina*
a
Faculty of Physics and International Laser Center, Lomonosov Moscow State University,
Moscow, 119991 Russia *Corresponding author: [email protected], tel. +79168208001
ABSTRACT The structural changes resulting from the reduction of disulphide bonds in three globular proteins (albumin, chymotrypsin and thrombin) in aqueous solutions are investigated using Raman spectroscopy. Changes in SS and SH vibrational bands are used for direct observation of the disulphide bond reduction. The corresponding changes in secondary structure and H-bonding of tyrosines are observed. It is shown that the unfolding pathway after the reduction of disulphide bridges in albumin depends on the reducing agent. The most developed changes are observed for the band assigned to the ggg conformation of the disulphide bridge in two similar serine proteases (thrombin and chymotrypsin) but the secondary structure of thrombin is much more stable against the reduction of the disulphide bonds.
Keywords: Raman spectroscopy; disulphide bridges; protein unfolding; serine proteases; serum albumins;
1. INTRODUCTION Disulphide bonds (bridges) between two cysteins of a polypeptide chain are important for protein stability and activity. CS–SC (), СС–SS (), and SS–CC () dihedral angles are used to characterize conformations of disulphide bridges. Three combinations of dihedral angles , , ≠ 0; 0, , ≠ 0; and , 0, ≠ 0 correspond to gauche-gauche-gauche (ggg), transgauche-gauche (tgg), and trans-gauche-trans (tgt) conformations, respectively [1–3].
1
Raman scattering spectroscopy is known to be a helpful tool in the study of protein structure in aqueous solutions. Raman bands peaking at 490–560 cm-1 are assigned to SS stretching mode of cystine in protein structure. Band positions 500–515, 520–530, and 540–550 cm-1 correspond to the ggg, tgg, and tgt conformations, respectively [1,4,5]. Amide I band (~1650 cm-1) is used to determine secondary structure of proteins [6–8]. Thrombin is a serine protease that plays the key role in blood coagulation, since it converts fibrinogen to fibrin in coagulation cascade. Bovine thrombin is a globular protein, consisting of two polypeptide chains (m.w. 37 kDa). The chains are linked by Cys1–Cys122 disulphide bond. The heavier chain, which is stabilized by Cys42–Cys58, Cys168–Cys182, and Cys191–Cys220 disulphide bridges, contains alpha-helical and beta-sheet fragments [9]. Cleavage of the four disulphide bonds leads to complete denaturation of the protein [10]. The Cys191–Cys220 bond controls the enzymatic activity of thrombin, since it determines the structure of the specificity pocket. The cleavage of the bond leads to a ~100-fold loss of the enzymatic activity [11]. Biochemical methods can hardly be used to comprehensively characterize changes of the remaining disulphide bonds that may indirectly affect the functional activity of thrombin [12]. Note insufficient data on the effect of disulphide bonds on thrombin functioning. Chymotrypsin is a well-studied serine protease (m.w. 25 kDa). Bovine chymotrypsin is a globular protein consisting of three polypeptide chains linked by Cys1–Cys122 and Cys136–Cys201 disulphide bridges. The chains are internally stabilized by Cys42–Cys58, Cys168–Cys182, and Cys191–Cys220 bonds. Beta sheets are dominant elements of chymotrypsin secondary structure [13,14]. The Cys191–Cys220 bond stabilizes the specificity pocket. The mutant enzyme without such a bridge exhibits a decrease in the enzymatic activity by several orders of magnitude [15]. Note similarity of the structural features of chymotrypsin and thrombin. In particular, four of five disulphide bridges in chymotrypsin (Cys1–Cys122, Cys42–Cys58, Cys168–Cys182, and Cys191– Cys220) are fully homologous to the four disulphide bonds of thrombin [16]. Thus, chymotrypsin can be compared with thrombin with respect to the role of disulphide bonds in stabilization of protein structure. Serum albumin is the main protein of blood plasma that provides unspecific bonding and transportation of fatty acids, steroids, and hormones in blood. It is widely employed as a model object in the study of the properties of globular proteins (m.w. 66.5 kDa). The polypeptide chain forms an asymmetric globule, in which α-helix is the dominant structure. The protein structure is stabilized by 17 disulphide bonds, six of which are located in the immediate vicinity of the binding sites [17]. The cleavage of all disulphide bonds in human serum albumin leads to 2
complete disorganization of tertiary structure [18]. Raman spectroscopy has been used to study conformational changes of disulphide bridges in BSA [19] and modification of α-helices due to cleavage of disulphide bonds [7]. The purposes of this work are the comparative study of the reduction of disulfide bonds in albumin, chymotrypsin, and thrombin in aqueous solutions using Raman spectroscopy, the analysis of the effect of the cleavage of disulphide bonds on the secondary structure and the Hbonding of tyrosine residues, and the study of the effect of two reducing agents on albumin. 2. MATERIALS AND METHODS 2.1. Samples In the experiments, we use lyophilized bovine thrombin (Sigma-Aldrich, CN T4648), lyophilized chymotrypsin (Samson-Med), and lyophilized BSA (MP Biomedicals, CN 160069). The proteins are dissolved in Milli-q water at a concentration of 100 mg/mL (1.5 mM for albumin, 3.8 mM for chymotrypsin, and 3.1 mM for thrombin) at pH 7.4. Native structure of thrombin, chymotrypsin, and albumin is not violated at pH 5–10 [20], 7–9 [21], and 5.0–8.0 [22], respectively. Dithiothreitol (DTT) at a concentration of 35 mM and tris(2carboxyethyl)phosphine (TCEP) at a concentration of 35 mM serve as reducing agents: Scheme 1. The DTT interaction with protein yields a reaction product (4,5-dihydroxy-1,2-dithiane (oxidized DTT)) that has an internal disulphide bond. For this reason, we do not use DTT in the experiments with thrombin and chymotrypsin. DTT is employed in the experiments with albumin for comparison with the available Raman data. Aqueous solution of NaOH is used for pH adjustment in the experiments with TCEP. The measurements are performed at room temperature immediately after adding reducing agents, and the evolution of spectra in time is analyzed. The experiments show the absence of variations in the spectra of pure proteins in time under laser irradiation in the course of measurements. 2.2. Raman measurements In the experiments, we use a high-sensitivity Raman spectrometer equipped with a Coherent Innova 90 cw argon-ion laser (excitation wavelength, 514 nm; power on the sample, up to 100 mW; spectral resolution, 2 cm-1; and measurement interval, 200–2000 cm-1 [23] ) and a ThermoScientific MicroRaman DXR Raman microscope (excitation wavelengths, 532 and 780 nm; excitation powers, 10 and 24 mW, respectively; spectral resolution, 2 cm-1; and 3
measurement interval, 40–3500 cm-1). The Raman microscope, which has a lower sensitivity, is used for simultaneous measurements of the Raman bands assigned to SS and SH stretching modes. To assess possible effects of laser radiation on the samples under study, we preliminary performed 4-hour measurements of the spectra of pure protein solutions at the highest laser power (100 mW). The absence of spectral changes in such measurements allows the spectroscopic analysis of changes caused by the cleavage of disulphide bonds. 2.3. Data processing A multistage procedure is used for data processing. First, the spectra are smoothed using the Savitsky-Golay filter [24] and the second derivatives of the spectral curves are calculated to eliminate the background contribution in the subsequent normalization procedure. Original spectra are normalized by the intensity of the band peaking at 1004–1006 cm-1 in the secondderivative spectrum. (The Raman band at 1004–1006 cm-1 is assigned to the breathing mode of the phenylalanine ring.) For the analysis of the amide I band, we perform normalization by the integral intensity of this band. The backgrounds are subtracted using a procedure of [25], which employs the iterative subtraction of the polynomial background. We use the fifth-power polynomials and separately perform background subtraction in intervals of 350–1030, 980–1800, and 2000–2800 cm-1. 3. RESULTS AND DISCUSSION First, we study Raman spectra of albumin in the presence of DTT that can be compared with the results of [7]. Figure 1. Figure 2. Figure 1a compares the spectra of albumin and albumin with DTT. It is seen that the quantitative analysis of the spectral changes is impeded due to significant contribution of the signal of oxidized DTT. Nevertheless, we observe a gradual decrease in the intensity of the bands of the disulphide bridges of the protein, which directly proves the reduction of disulphide bonds (Figure 1b). Note that the reduction takes place for all basic conformations of the bridges. A detailed analysis of the spectral changes is possible with the aid of spectral deconvolution [7]. The cleavage of disulphide bonds is accompanied by minor variations in the relative intensities of the tyrosine doublet (I850/I830) and a shift of the high-frequency component of the doublet (Fig. 2a). Such spectral changes indicate modification of the H-bonds of the tyrosine residues [8,26,27] and, hence, conformational changes of the protein molecule. 4
Note variations in the intensities of the bands assigned to CC skeletal vibrations of the polypeptide change (Fig. 2b). A decrease in the intensity of the band at 940 cm-1 upon thermal denaturation has been observed in [28] and interpreted as a decrease in the amount of -helices. The minor changes in a spectral interval of 990–1000 cm-1 are presumably due to the contribution of oxidized DTT signal. The spectral changes in the range of the amide I band (Fig. 2c) correspond to degradation of the secondary structure, since the high-frequency shift of the band and a decrease in the intensity can be interpreted as a decrease in the amount of -helices and an increase in the amount of randomcoil fragments. Similar changes of the amide I band have been observed in [7]. Figure 3. Figure 3 compares the spectra of albumin and albumin with TCEP. A decrease in the intensity of Raman band at 500 cm-1 proves the dominant cleavage of disulphide bonds in the ggg conformation (Fig. 3a). A minor increase in the signal intensity at frequencies corresponding to the tgg and tgt conformations may indicate that the cleavage of the SS bonds in the ggg conformation is accompanied by partial transformation into less stable tgg and tgt conformations. As in the case of interaction with DTT, cleavage and transformation of disulfide bonds leads to modification of the H-bonds of tyrosine residues , which follows from intensity variations of the tyrosine doublet and a shift of the band peaking at 850 cm-1 (Fig. 3b). We also observe a shift of the amide I band and a decrease in the intensity at 1650 cm-1 (Fig. 3c). Changes of the band assigned to the CC skeletal vibrations can hardly be analyzed, since the corresponding Raman band is overlapped with the TCEP band. After 3 h of interaction with TCEP, the solution becomes turbid presumably due to aggregation of albumin and a significant increase in the background signal impedes Raman measurements. Figure 4. TCEP causes reduction of disulfide bonds of chymotrypsin, and (as in the case of albumin) the most developed spectral changes are observed at frequencies corresponding to the ggg conformation (Fig. 4a). The cleavage of disulfide bonds is additionally proven by an increase in the intensity of the Raman signal corresponding to the SH stretching mode (about 2580 cm-1, Fig. 4c) [29]. Raman band at 2450 cm-1 in the samples with TCEP may correspond to the PH stretching mode of TCEP molecule [30].
5
Changes of the secondary structure resulting from the cleavage of disulfide bonds lead to spectral changes in the range of the amide I band (Fig. 4b). We observe a decrease in the intensity at 1670 cm-1 and an increase in the intensity of the high-frequency components that can be interpreted as partial transformation of sheets into random coils. Beta structure of chymotrypsin is stabilized by three disulphide bonds in the ggg (Cys42–Cys58 and Cys136–Cys201) and tgt (Cys168–Cys182) conformations [13,14]). Apparently, the observed spectral changes are due to cleavage of such bonds. Figure 5. Fig. 5a shows a significant decrease in the signal intensity in the spectral interval of the SS bonds and a corresponding increase in the intensity of the band assigned to the SH stretching mode. The strongest spectral changes are observed in the interval of the disulphide bridges in the ggg conformation as in experiments with chymotrypsin. Again, a minor increase in the signal intensity at frequencies corresponding to the tgg conformation may indicate that the cleavage of the SS bonds in the ggg conformation is accompanied by partial transformation into less stable tgg conformation. Consistent changes are observed in the SH vibration region (Fig. 5c). In comparison with chymotrypsin, thrombin exhibits smaller changes of the amide I band that can be interpreted as partial transformation of helices into sheets (Fig. 5b). Based on the Raman data, we may assume that the structure of thrombin is more stable against cleavage of disulfide bonds. The X-ray data show that two of four disulfide bridges in thrombin have ggg conformation (Cys168–Cys182 and Cys122–Cys1 (interchain)). The results of [10] show that the Cys168–Cys182 bond is the first to be reduced due to interaction with DTT. The cleavage of this bond may lead to changes of the α-helical and β-sheet fragments containing Cys168 and Cys182, respectively. Such changes are in agreement with the results of Fig. 5b. Minor changes in the relative intensities of the tyrosine doublet in the experiments with thrombin and chymotrypsin indicate insignificant changes of the corresponding H-bonds due to the cleavage of disulphide bonds in the two proteins. 4. CONCLUSIONS The cleavage of disulfide bonds in proteins in aqueous solutions can be monitored using variations in the intensities of the Raman bands assigned to the SS and SH stretching modes. The experiments with albumin (evolution of the bands assigned to disulphide bridges and tyrosine doublet) show that the application of different reducing agents leads to different unfolding 6
pathways. The known decrease in the catalytic activity of chymotrypsin related to the cleavage of disulphide bonds may result from both direct changes of the active site and noticeable modification of the secondary structure. Based on the results for the three proteins, we may conclude that disulfide bridges control conformational stability of each protein molecule as a whole and the cleavage of disulfide bonds may lead to less ordered structures with greater contents of random coils. The most developed changes in the spectra of two serine proteases (chymotrypsin and thrombin) in the presence of TCEP are observed at frequencies corresponding to the ggg conformation. Note that the cleavage of disulphide bridges in the serine proteases causes substantially different changes of the secondary structure.
ACKNOWLEDGMENTS We are grateful to A.V. Golovin, Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University for helpful discussions. This work was supported in part by the Russian Foundation for Basic Research (project no. 15-02-06512) and Program for Development of Moscow State University.
REFERENCES [1] H. Sugeta, A. Go, T. Miyazawa, Vibrational Spectra and Molecular Conformations of Dialkyl Disulfides, Bull. Chem. Soc. Jpn. 46 (1973) 3407–3411. doi:10.1246/bcsj.46.3407. [2] W. Zhao, S. Krimm, Vibrational studies of the disulfide group in proteins: Part II. Ab initio force fields and normal mode frequencies of dimethyl, methylethyl, and diethyl disulfides, J. Mol. Struct. 224 (1990) 7–20. doi:10.1016/0022-2860(90)87003-G. [3] C.H. Görbitz, Conformational properties of disulphide bridges. 2. Rotational potentials of diethyl disulphide, J. Phys. Org. Chem. 7 (1994) 259–267. [4] H.E. Van Wart, H.A. Scheraga, Agreement with the disulfide stretching frequencyconformation correlation of Sugeta, Go, and Miyazawa, Proc. Natl. Acad. Sci. 83 (1986) 3064–3067. [5] W. Qian, S. Krimm, Vibrational studies of the disulfide group in proteins. Part V. Correlation of SS stretch frequencies with the CCSS dihedral angle in known protein disulfide bridges, Biopolymers. 32 (1992) 321–326. [6] J.T. Pelton, L.R. McLean, Spectroscopic Methods for Analysis of Protein Secondary Structure, Anal. Biochem. 277 (2000) 167–176. doi:10.1006/abio.1999.4320.
7
[7] C. David, S. Foley, C. Mavon, M. Enescu, Reductive unfolding of serum albumins uncovered by Raman spectroscopy, Biopolymers. 89 (2008) 623–634. doi:10.1002/bip.20972. [8] D. Nemecek, J. Stepanek, G.J. Thomas, Raman Spectroscopy of Proteins and Nucleoproteins, in: J.E. Coligan, B.M. Dunn, D.W. Speicher, P.T. Wingfield (Eds.), Curr. Protoc. Protein Sci., John Wiley & Sons, Inc., Hoboken, NJ, USA, 2013. [9] B.J. Desai, R.S. Boothello, A.Y. Mehta, J.N. Scarsdale, H.T. Wright, U.R. Desai, Interaction of thrombin with sucrose octasulfate, Biochemistry (Mosc.). 50 (2011) 6973– 6982. doi:10.1021/bi2004526. [10] R. Rajesh Singh, J.-Y. Chang, Structural stability of human α-thrombin studied by disulfide reduction and scrambling, Biochim. Biophys. Acta BBA - Proteins Proteomics. 1651 (2003) 85–92. doi:10.1016/S1570-9639(03)00238-3. [11] L.A. Bush-Pelc, F. Marino, Z. Chen, A.O. Pineda, F.S. Mathews, E. Di Cera, Important Role of the Cys-191 Cys-220 Disulfide Bond in Thrombin Function and Allostery, J. Biol. Chem. 282 (2007) 27165–27170. doi:10.1074/jbc.M703202200. [12] A.A. Serejskaya, S.V. Zubak, T.W. Osadchuk, S.B. Serebryani, Enzymic activity of thrombin with partially reduced disulfide bonds, Thromb. Res. 32 (1983) 147–154. [13] K. Brady, A.Z. Wei, D. Ringe, R.H. Abeles, Structure of chymotrypsin-trifluoromethyl ketone inhibitor complexes: comparison of slowly and rapidly equilibrating inhibitors, Biochemistry (Mosc.). 29 (1990) 7600–7607. [14] A. Razeto, B. Galunsky, V. Kasche, K.S. Wilson, V.S. Lamzin, High resolution structure of native bovine alpha-chymotrypsin, Be Publ. (n.d.). doi:10.2210/pdb1yph/pdb. [15] Varallyay E., Lengyel Z., Graf L., Szilagyi L., The Role of Disulfide Bond C191-C220 in Trypsin and Chymotrypsin, BBRC. (1997) 592–596. [16] R.W. Colman, Hemostasis and Thrombosis: Basic Principles and Clinical Practice, Lippincott Williams & Wilkins, 2006. [17] K.A. Majorek, P.J. Porebski, A. Dayal, M.D. Zimmerman, K. Jablonska, A.J. Stewart, M. Chruszcz, W. Minor, Structural and immunologic characterization of bovine, horse, and rabbit serum albumins, Mol. Immunol. 52 (2012) 174–182. doi:10.1016/j.molimm.2012.05.011. [18] G. Markus, F. Karush, The Disulfide Bonds of Human Serum Albumin and Bovine γGlobulin1, J. Am. Chem. Soc. 79 (1957) 134–139. [19] K. Nakamura, S. Era, Y. Ozaki, M. Sogami, T. Hayashi, M. Murakami, Conformational changes in seventeen cystine disulfide bridges of bovine serum albumin proved by Raman spectroscopy, FEBs Lett. 417 (1997) 375–378. [20] R. Machovich, Thrombin The, CRC-Press, 1984. [21] H.U. Bergmeyer, K. Gawehn, Methods of enzymatic analysis, Verlag Chemie, 1974. [22] T. Peters Jr, All about albumin: biochemistry, genetics, and medical applications, Academic press, 1995. [23] N.N. Brandt, A.Y. Chikishev, Laser Raman spectrometer for the studies of biomolecules with monitoring temperature of the samples, Laser Phys. 12 (2002) 647–652. [24] A. Savitzky, M.J.E. Golay, Smoothing and Differentiation of Data by Simplified Least Squares Procedures., Anal. Chem. 36 (1964) 1627–1639. doi:10.1021/ac60214a047. 8
[25] C.A. Lieber, A. Mahadevan-Jansen, Automated method for subtraction of fluorescence from biological Raman spectra, Appl. Spectrosc. 57 (2003) 1363–1367. [26] M.N. Siamwiza, R.C. Lord, M.C. Chen, T. Takamatsu, I. Harada, H. Matsuura, T. Shimanouchi, Interpretation of the doublet at 850 and 830 cm-1 in the Raman spectra of tyrosyl residues in proteins and certain model compounds, Biochemistry (Mosc.). 14 (1975) 4870–4876. [27] J.L. McHale, Fermi resonance of tyrosine and related compounds. Analysis of the Raman doublet, J. Raman Spectrosc. 13 (1982) 21–24. [28] V.J.C. Lin, J.L. Koenig, Raman studies of bovine serum albumin, Biopolymers. 15 (1976) 203–218. doi:10.1002/bip.1976.360150114. [29] H. Li, G.J. Thomas, Studies of virus structure by Raman spectroscopy. Cysteine conformation and sulfhydryl interactions in proteins and viruses. 1. Correlation of the Raman sulfur-hydrogen band with hydrogen bonding and intramolecular geometry in model compounds, J. Am. Chem. Soc. 113 (1991) 456–462. doi:10.1021/ja00002a012. [30] N.B. Colthup, L.H. Daly, S.E. Wiberley, Introduction to Infrared and Raman Spectroscopy, Elsevier, 1990.
9
FIGURE AND SCHEME CAPTIONS Figure 1. (a) Raman spectra (ex = 514.5 nm) of (solid line) albumin in aqueous solution and (dashed line) oxidized DTT and (b) spectra of albumin solutions with DTT measured over three subsequent time intervals of 80 min.
10
Figure 2. (Upper curves) Raman spectra (ex = 514.5 nm) of (black line) albumin aqueous solution and albumin solutions with DTT measured over time intervals of (dark gray line) 0–80, (grey line) 80–160, and (light grey line) 160–240 min. and (lower curves) scaled-up differences of the above spectra and the spectrum of albumin aqueous solution.
11
Figure 3. (Upper curves) Raman spectra (ex = 514.5 nm) of (black line) albumin aqueous solution and albumin solutions with TCEP measured over time intervals of (dark gray line) 0–40, (grey line) 40–80, (light grey line) 80–120, and (dashed line) 120–160 min. and (lower curves) scaled-up differences of the above spectra and the spectrum of albumin aqueous solution.
12
Figure 4. (Upper curves) Raman spectra (ex = 532 nm) of (black line) chymotrypsin aqueous solution and albumin solutions with TCEP measured over time intervals of (dark gray line) 40– 80, (grey line) 120–160, and (light grey line) 200–240 min. and (lower curves) scaled-up differences of the above spectra and the spectrum of albumin aqueous solution.
13
Figure 5. (Upper curves) Raman spectra (ex = 780 nm) of (black line) thrombin aqueous solution and albumin solutions with TCEP measured over time intervals of (grey line) 0–120, and (dashed line) 120–240 min. and (lower curves) scaled-up differences of the above spectra and the spectrum of albumin aqueous solution.
14
Scheme 1. Reduction of protein disulphide bonds with DTT and TCEP.
15