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Overexpression, purification, and enthalpy of unfolding of ferricytochrome c552 from a psychrophilic microorganism
Journal of Inorganic Biochemistry 131 (2014) 76–78
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio
Short communication
Overexpression, purification, and enthalpy of unfolding of ferricytochrome c552 from a psychrophilic microorganism Victoria F. Oswald a, WeiTing Chen a, Paul B. Harvilla a,b, John S. Magyar a,⁎ a b
Department of Chemistry, Barnard College, Columbia University, New York, NY 10027, United States Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States
a r t i c l e
i n f o
Article history: Received 28 May 2013 Received in revised form 4 November 2013 Accepted 5 November 2013 Available online 12 November 2013 Keywords: Psychrophile Cytochrome c552 Van't Hoff enthalpy Thermal denaturation
a b s t r a c t The psychrophilic, hydrocarbonoclastic microorganism Colwellia psychrerythraea is important in global nutrient cycling and bioremediation. In order to investigate how this organism can live so efficiently at low temperatures (~4 °C), thermal denaturation studies of a small electron transfer protein from Colwellia were performed. Colwellia cytochrome c552 was overexpressed in Escherichia coli, isolated, purified, and characterized by UV–visible absorption spectroscopy. The melting temperature (Tm) and the van't Hoff enthalpy (ΔHvH) were determined. These values suggest an unexpectedly high stability for this psychrophilic cytochrome.
Here, we report the first studies of cytochrome c552 from the psychrophilic microorganism Colwellia psychrerythraea. We describe its overexpression, purification, UV–vis absorption spectrum, and enthalpy of unfolding determined by thermal denaturation. In contrast with the extensive literature on proteins from thermophilic organisms, only a few proteins from psychrophilic organisms have been characterized to date, with few consistent broad conclusions [1–8]. Elucidating the molecular basis of psychrophilicity is important for expanding fundamental understanding of protein folding dynamics and biological electron transfer processes. Psychrophilic organisms such as C. psychrerythraea play essential roles in global nutrient cycling and bioremediation; Colwellia species were a major component of the microbial community response in the deep, cold waters of the Gulf of Mexico following the 2010 Deepwater Horizon oil spill [9]. The published genome of the hydrocarbonoclastic (hydrocarbon-degrading), obligately psychrophilic (cold-loving) bacterium C. psychrerythraea 34H reveals the presence of a gene (gene ID 637282778) coding for a homolog of the electron-transfer protein cytochrome c552 (Cpcyt c552) [10]. The amino acid sequence is provided in the Supplementary Information (Fig. S1). The structure is predicted to be that of a globular cytochrome; its closest match in a BLAST search of the PDB is cyt c 552 from the mesophilic marine
⁎ Corresponding author. Tel.: +1 212 854 2074; fax: +1 212 854 2310. E-mail address: [email protected] (J.S. Magyar). 0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2013.11.002
© 2013 Elsevier Inc. All rights reserved.
bacterium Marinobacter hydrocarbonoclasticus (formerly known as Pseudomonas nautica) [11–13]. The structure, folding, and dynamics of globular cytochromes c from a wide variety of mesophilic [14–17] and thermophilic [18–22] organisms have been well studied, making this protein an excellent model system for an examination of psychrophilic adaptations. The bright red color and unmistakable iron porphyrin absorption spectrum aid in isolation, purification, and characterization of cytochrome c; and since the heme group is covalently bound to the protein through a conserved Cys-X-X-Cys-His motif, the heme remains attached to the amino acid chain even under denaturing conditions. A synthetic gene for Cpcyt c552 was co-transformed into Escherichia coli with the pEC86 heme cassette [23] to overexpress a properly folded cytochrome with a covalently bound heme. Experimental details describing cloning, protein overexpression, and protein purification are provided in the Supplementary information. Spectroscopic experiments are performed in 100 mM sodium phosphate buffer, pH 7.0, in septumtopped quartz cuvettes using a Varian Cary 5000 UV–vis-NIR spectrophotometer with a Peltier temperature-controlled sample compartment. The sample compartment and optics are purged with a flow of dry dinitrogen gas to prevent water condensation on the cuvette at low temperatures. The mass of purified Cpcyt c552 has been confirmed by MALDI-TOF mass spectrometry (Scripps Center for Mass Spectrometry, La Jolla, CA); calculated mass 8832 Da, measured m/z 8834. The N-terminal signal peptide is correctly cleaved in E. coli and does not appear in the purified protein. The UV–visible absorption spectrum of FeIIICpcyt c552 is shown in Fig. 1. The primary features are the Soret
V.F. Oswald et al. / Journal of Inorganic Biochemistry 131 (2014) 76–78
Fig. 1. UV–visible absorption spectrum of ferricytochrome c552 from Colwellia psychrerythraea in sodium phosphate buffer, pH 7.0. The inset highlights the band at 698 nm attributed to Fe–methionine(S) coordination.
band (ε ~ 104,000 M−1 cm−1) at 411 nm, and the Q-bands between 500 and 600 nm, with a maximum at 525 nm (ε ~ 9500 M−1 cm−1). Also visible in the spectrum of FeIIICpcyt c552 is the band at 698 nm that is attributed to Fe–S(methionine) coordination (inset, Fig. 1) [24,25]. The shape, intensity, and position of these bands are commonly used as a probe of protein folding. Since we are most interested in thermal effects on protein folding for this psychrophilic protein, we have used heat to denature Cpcyt c552, monitoring the unfolding by UV–vis absorption spectroscopy (Fig. 2). Spectra were collected every 2 K from 275 to 359 K (2–86 °C), with a 10-minute temperature equilibration period between each measurement. The thermal ramp and spectral data collection are computer controlled using software provided by Agilent. Upon heating and protein denaturation, the Soret band shifts to higher energy and the Q-bands broaden and shift. The FeIII–S(Met) band at 698 nm in the oxidized protein disappears upon heating, indicating the loss of heme Fe–methionine coordination when the protein unfolds. The thermal denaturation of Cpcyt c552 is irreversible. For the current analyses, we assume that the Lumry–Eyring model holds, i.e., that reversible protein unfolding is followed by irreversible changes in the unfolded protein at high temperatures [26]. A thermal denaturation curve can be produced by plotting the absorbance at 411 nm (λmax of the Soret band for the folded protein) vs. temperature. Using the method of John and Weeks [27], we have fit the first derivative of each thermal denaturation curve to a modified form of the van't Hoff equation. Fitting the first derivative rather than the raw data minimizes the effect of the baseline on the fitting results [27,28]. From these fits, we have determined the van't Hoff enthalpy (ΔHvH) and the melting temperature (Tm) for Cpcyt c552 (Table 1). Since at Tm, ΔG = 0 and ΔS = ΔHvH/Tm, we can also calculate the unfolding entropy ΔS for this protein (Table 1). Limited enthalpy data are available in the literature for thermal denaturation of cyt c at neutral pH [29,30]; here we will compare to ferricytochromes c from the mesophilic eukaryotes yeast and horse and the thermophilic bacterium Thermus thermophilus.1
1 Table S1 presents a multiple sequence alignment for Cpcyt c552 with cyt c from these organisms. Although the sequence identities with Cpcyt c552 are low (15.7–20.6 %), the structures of these cytochromes are all very similar to that of the predicted structure of Cpcyt c552. Also included in the alignment is the sequence of cyt c552 from the mesophile M. hydrocarbonoclasticus, the closest match to Cpcyt c552 in the PDB.
77
For yeast iso-1-cytochrome c, a plot of ΔHvH vs. Tm is linear in the range of pH 3–5 [31,32]. Extrapolation to pH 7 yields ΔH vH = 398 kJ/mol and T m = 333 K (Table 1). It is of note that Cpcyt c 552 has a T m that is identical (within error, 334 ± 2 K) to that of yeast iso-1-cytochrome c at pH 7, while ΔH vH for Cpcyt c 552 is lower by ~ 90 kJ/mol than that of yeast iso-1-cytochrome c (ΔHvH = 309 ± 33 kJ/mol). In the case of horse heart cytochrome c, both Tm and ΔHvH are high [33]. By contrast, a thermophilic cyt c from T. thermophilus has both a higher Tm and a significantly lower ΔHvH (Table 1) [34]. One might expect that if a thermophilic cyt c is characterized by lower ΔH and ΔS and higher Tm values when compared to a mesophilic homolog, then psychrophilic proteins would be characterized by lower Tm, and increased values of ΔH and ΔS. The results reported here, however, indicate that this is not the case. Our findings reveal that psychrophilic Cpcyt c552 has a lower ΔHvH and unchanged Tm compared to similar mesophilic proteins and suggest that there exist mechanisms of protein stabilization in the psychrophile that are unexpected based on sequence alone. It will be important to examine the three-dimensional structure of the protein for insights into the molecular basis of this stabilization. In particular, it has been suggested that psychrophilic proteins have decreased intramolecular interactions (e.g., hydrogen bonds) in order to increase flexibility, with an expected concomitant decrease in stability [6,35,36]. The large number of alanines in the amino acid sequence of Cpcyt c552 (16 residues, 20.3%) is consistent with the prediction of greater protein flexibility. The relatively short, hydrophobic alanine side chain should decrease steric conflicts and increase overall protein chain flexibility, as demonstrated by the intrachain diffusion studies of Kiefhaber and coworkers [37,38]. Our work suggests that the predicted flexibility of psychrophilic Cpcyt c552 does not correlate with overall protein stability toward unfolding. Protein dynamics (flexibility) and protein stability are not simply coupled in this protein. Further calorimetric, spectroscopic, and structural studies to investigate these issues are underway. Abbreviations wavelength of maximum absorbance λmax Cpcyt c552 Colwellia psychrerythraea cytochrome c552 ΔHvH van't Hoff enthalpy IPTG isopropyl β-D-1-thiogalactopyranoside melting temperature Tm
Acknowledgments We thank Linda Thöny-Meyer for generously making the pEC86 plasmid available. We gratefully acknowledge the funding from a Camille and Henry Dreyfus Foundation Faculty Start-Up Award, a Barnard College Presidential Research Award, the National Science Foundation (MRI-R2 CHE-0959177 and ARI-R2 CHE-0961709), the New York Division of Science, Technology and Innovation (NYSTAR), the Columbia NSF NSEC: Center for Electron Transport in Molecular Nanostructures (CHE-0117752 and CHE-0641523), a grant to Barnard College from the Undergraduate Science Education Program of the Howard Hughes Medical Institute (VFO), the Barnard College Con Edison Summer Internship in Science Program (WC), the NIH Training Program in Molecular Biophysics at Columbia (PBH) (T32 GM008281), and Barnard College. No funding sources had any role in the conduct of the research or preparation of the report. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jinorgbio.2013.11.002.
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V.F. Oswald et al. / Journal of Inorganic Biochemistry 131 (2014) 76–78
Fig. 2. Thermal denaturation of ferricytochrome c552 from Colwellia psychrerythraea (sodium phosphate buffer, pH 7.0), monitored by UV–vis absorption spectroscopy.
Table 1 Thermodynamic parameters for ferricytochrome c unfolding. Protein
ΔHvH (kJ/mol)
Tm (K)
ΔS (kJ/mol K)
Reference
C. psychrerythraea cyt c552a Yeast iso-1-cyt cb Horse heart cyt cc T. thermophilus cyt c552d
309 ± 33 398 410 130
334 ± 2 333 357 389
0.925 1.20 1.15 0.334
This study [31,32] [33] [34]
a b c d
Determined spectroscopically; Soret band. Data reported ±95% confidence level. Determined spectroscopically; Soret band. Extrapolated from low pH data. Determined by differential scanning calorimetry. Determined spectroscopically; methionine ligation.
References [1] Z.-X. Liang, I. Tsigos, T. Lee, V. Bouriotis, K.A. Resing, N.G. Ahn, J.P. Klinman, Biochemistry 43 (2004) 14676–14683. [2] S. DasSarma, M.D. Capes, R. Karan, P. DasSarma, PLoS ONE 8 (2013) e58587. [3] S. Yamauchi, Y. Ueda, M. Matsumoto, U. Inoue, H. Hayashi, Extremophiles 16 (2012) 871–882. [4] Y. Sato, S. Watanabe, N. Yamaoka, Y. Takada, Extremophiles 12 (2008) 107–117. [5] H.-K.S. Leiros, A.L. Pey, M. Innselset, E. Moe, I. Leiros, I.H. Steen, A. Martinez, J. Biol. Chem. 282 (2007) 21973–21986. [6] R.A. Goldstein, Prot. Sci. 16 (2007) 1887–1895. [7] G. Di Rocco, G. Battistuzzi, C.A. Bortolotti, M. Borsari, E. Ferrari, S. Monari, M. Sola, J. Biol. Inorg. Chem. 16 (2011) 461–471. [8] K.S. Siddiqui, T.J. Williams, D. Wilkins, S. Yau, M.A. Allen, M.V. Brown, F.M. Lauro, R. Cavicchioli, Annu. Rev. Earth Planet. Sci. 41 (2013) 6.1–6.29. [9] M.C. Redmond, D.L. Valentine, Proc. Natl. Acad. Sci. U. S. A. 109 (2011) 20292–20297. [10] B.A. Methé, K.E. Nelson, J.W. Deming, B. Momen, E. Melamud, X. Zhang, J. Moult, R. Madupu, W.C. Nelson, R.J. Dodson, L.M. Brinkac, S.C. Daugherty, A.S. Durkin, R.T. DeBoy, J.F. Kolonay, S.A. Sullivan, L. Zhou, T.M. Davidsen, M. Wu, A.L. Huston, M. Lewis, B. Weaver, J.F. Weidman, H. Khouri, T.R. Utterback, T.V. Feldblyum, C.M. Fraser, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 10913–10918.
[11] K. Brown, D. Nurizzo, S. Besson, W. Shepard, J. Moura, I. Moura, M. Tegoni, C. Cambillau, J. Mol. Biol. 289 (1999) 1017–1028. [12] C. Spröer, E. Lang, P. Hobeck, J. Burghardt, E. Stackebrandt, B.J. Tindall, Int. J. Syst. Bacteriol. 48 (1998) 1445–1448. [13] S.F. Altschul, T.L. Madden, A.A. Schäffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Nucleic Acids Res. 25 (1997) 3389–3402. [14] E.V. Pletneva, H.B. Gray, J.R. Winkler, J. Mol. Biol. 345 (2004) 855–867. [15] J.G. Lyubovitsky, H.B. Gray, J.R. Winkler, J. Am. Chem. Soc. 124 (2002) 5481–5485. [16] M.C.R. Shastry, J.M. Sauder, H. Roder, Acc. Chem. Res. 31 (1998) 717–725. [17] S.W. Englander, T.R. Sosnick, L.C. Mayne, M. Shtilerman, P.X. Qi, Y. Bai, Acc. Chem. Res. 31 (1998) 737–744. [18] K.L. Bren, J.A. Kellogg, R. Kaur, X. Wen, Inorg. Chem. 43 (2004) 7934–7944. [19] X. Wen, K.L. Bren, Biochemistry 44 (2005) 5225–5233. [20] L. Zhong, X. Wen, T.M. Rabinowitz, B.S. Russell, E.F. Karan, K.L. Bren, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 8637–8642. [21] J. Hasegawa, S. Uchiyama, Y. Tanimoto, M. Mizutani, Y. Kobayashi, Y. Sambongi, Y. Igarashi, J. Biol. Chem. 275 (2000) 37824–37828. [22] S. Nakamura, S.-i. Ichiki, H. Takashima, S. Uchiyama, J. Hasegawa, Y. Kobayashi, Y. Sambongi, T. Ohkubo, Biochemistry 45 (2006) 6115–6123. [23] L. Thöny-Meyer, Biochemistry 42 (2003) 13099–13105. [24] A. Schejter, P. George, Biochemistry 3 (1964) 1045–1049. [25] W.A. Eaton, R.M. Hochstrasser, J. Chem. Phys. 46 (1967) 2533–2539. [26] R. Lumry, H. Eyring, J. Phys. Chem. 58 (1954) 110–120. [27] D.M. John, K.M. Weeks, Prot. Sci. 9 (2000) 1416–1419. [28] D.L. Allen, G.J. Pielak, Prot. Sci. 7 (1998) 1262–1263. [29] W. Pfeil, Protein Stability and Folding: A Collection of Thermodynamic Data, Springer, New York, 1998. [30] W. Pfeil, Protein Stability and Folding: A Collection of Thermodynamic Data, Supplement 1, Springer, New York, 2001. [31] L.M. Herrmann, B.E. Bowler, Prot. Sci. 6 (1997) 657–665. [32] D.S. Cohen, G.J. Pielak, Prot. Sci. 3 (1994) 1253–1260. [33] J. Bágel'ová, M. Antalík, M. Bona, Biochem. J. 297 (1994) 99–101. [34] K. Hon-nami, T. Oshima, Biochemistry 18 (1979) 5693–5697. [35] C. Struvay, G. Feller, Int. J. Mol. Sci. 13 (2012) 11643–11665. [36] S. D'Amico, T. Collins, J.-C. Marx, G. Feller, C. Gerday, EMBO Rep. 7 (2006) 385–389. [37] F. Krieger, B. Fierz, O. Bieri, M. Drewello, T. Kiefhaber, J. Mol. Biol. 332 (2003) 265–274. [38] O. Bieri, J. Wirz, B. Hellrung, M. Schutkowski, M. Drewello, T. Kiefhaber, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 9597–9601.
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio
Short communication
Overexpression, purification, and enthalpy of unfolding of ferricytochrome c552 from a psychrophilic microorganism Victoria F. Oswald a, WeiTing Chen a, Paul B. Harvilla a,b, John S. Magyar a,⁎ a b
Department of Chemistry, Barnard College, Columbia University, New York, NY 10027, United States Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States
a r t i c l e
i n f o
Article history: Received 28 May 2013 Received in revised form 4 November 2013 Accepted 5 November 2013 Available online 12 November 2013 Keywords: Psychrophile Cytochrome c552 Van't Hoff enthalpy Thermal denaturation
a b s t r a c t The psychrophilic, hydrocarbonoclastic microorganism Colwellia psychrerythraea is important in global nutrient cycling and bioremediation. In order to investigate how this organism can live so efficiently at low temperatures (~4 °C), thermal denaturation studies of a small electron transfer protein from Colwellia were performed. Colwellia cytochrome c552 was overexpressed in Escherichia coli, isolated, purified, and characterized by UV–visible absorption spectroscopy. The melting temperature (Tm) and the van't Hoff enthalpy (ΔHvH) were determined. These values suggest an unexpectedly high stability for this psychrophilic cytochrome.
Here, we report the first studies of cytochrome c552 from the psychrophilic microorganism Colwellia psychrerythraea. We describe its overexpression, purification, UV–vis absorption spectrum, and enthalpy of unfolding determined by thermal denaturation. In contrast with the extensive literature on proteins from thermophilic organisms, only a few proteins from psychrophilic organisms have been characterized to date, with few consistent broad conclusions [1–8]. Elucidating the molecular basis of psychrophilicity is important for expanding fundamental understanding of protein folding dynamics and biological electron transfer processes. Psychrophilic organisms such as C. psychrerythraea play essential roles in global nutrient cycling and bioremediation; Colwellia species were a major component of the microbial community response in the deep, cold waters of the Gulf of Mexico following the 2010 Deepwater Horizon oil spill [9]. The published genome of the hydrocarbonoclastic (hydrocarbon-degrading), obligately psychrophilic (cold-loving) bacterium C. psychrerythraea 34H reveals the presence of a gene (gene ID 637282778) coding for a homolog of the electron-transfer protein cytochrome c552 (Cpcyt c552) [10]. The amino acid sequence is provided in the Supplementary Information (Fig. S1). The structure is predicted to be that of a globular cytochrome; its closest match in a BLAST search of the PDB is cyt c 552 from the mesophilic marine
⁎ Corresponding author. Tel.: +1 212 854 2074; fax: +1 212 854 2310. E-mail address: [email protected] (J.S. Magyar). 0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2013.11.002
© 2013 Elsevier Inc. All rights reserved.
bacterium Marinobacter hydrocarbonoclasticus (formerly known as Pseudomonas nautica) [11–13]. The structure, folding, and dynamics of globular cytochromes c from a wide variety of mesophilic [14–17] and thermophilic [18–22] organisms have been well studied, making this protein an excellent model system for an examination of psychrophilic adaptations. The bright red color and unmistakable iron porphyrin absorption spectrum aid in isolation, purification, and characterization of cytochrome c; and since the heme group is covalently bound to the protein through a conserved Cys-X-X-Cys-His motif, the heme remains attached to the amino acid chain even under denaturing conditions. A synthetic gene for Cpcyt c552 was co-transformed into Escherichia coli with the pEC86 heme cassette [23] to overexpress a properly folded cytochrome with a covalently bound heme. Experimental details describing cloning, protein overexpression, and protein purification are provided in the Supplementary information. Spectroscopic experiments are performed in 100 mM sodium phosphate buffer, pH 7.0, in septumtopped quartz cuvettes using a Varian Cary 5000 UV–vis-NIR spectrophotometer with a Peltier temperature-controlled sample compartment. The sample compartment and optics are purged with a flow of dry dinitrogen gas to prevent water condensation on the cuvette at low temperatures. The mass of purified Cpcyt c552 has been confirmed by MALDI-TOF mass spectrometry (Scripps Center for Mass Spectrometry, La Jolla, CA); calculated mass 8832 Da, measured m/z 8834. The N-terminal signal peptide is correctly cleaved in E. coli and does not appear in the purified protein. The UV–visible absorption spectrum of FeIIICpcyt c552 is shown in Fig. 1. The primary features are the Soret
V.F. Oswald et al. / Journal of Inorganic Biochemistry 131 (2014) 76–78
Fig. 1. UV–visible absorption spectrum of ferricytochrome c552 from Colwellia psychrerythraea in sodium phosphate buffer, pH 7.0. The inset highlights the band at 698 nm attributed to Fe–methionine(S) coordination.
band (ε ~ 104,000 M−1 cm−1) at 411 nm, and the Q-bands between 500 and 600 nm, with a maximum at 525 nm (ε ~ 9500 M−1 cm−1). Also visible in the spectrum of FeIIICpcyt c552 is the band at 698 nm that is attributed to Fe–S(methionine) coordination (inset, Fig. 1) [24,25]. The shape, intensity, and position of these bands are commonly used as a probe of protein folding. Since we are most interested in thermal effects on protein folding for this psychrophilic protein, we have used heat to denature Cpcyt c552, monitoring the unfolding by UV–vis absorption spectroscopy (Fig. 2). Spectra were collected every 2 K from 275 to 359 K (2–86 °C), with a 10-minute temperature equilibration period between each measurement. The thermal ramp and spectral data collection are computer controlled using software provided by Agilent. Upon heating and protein denaturation, the Soret band shifts to higher energy and the Q-bands broaden and shift. The FeIII–S(Met) band at 698 nm in the oxidized protein disappears upon heating, indicating the loss of heme Fe–methionine coordination when the protein unfolds. The thermal denaturation of Cpcyt c552 is irreversible. For the current analyses, we assume that the Lumry–Eyring model holds, i.e., that reversible protein unfolding is followed by irreversible changes in the unfolded protein at high temperatures [26]. A thermal denaturation curve can be produced by plotting the absorbance at 411 nm (λmax of the Soret band for the folded protein) vs. temperature. Using the method of John and Weeks [27], we have fit the first derivative of each thermal denaturation curve to a modified form of the van't Hoff equation. Fitting the first derivative rather than the raw data minimizes the effect of the baseline on the fitting results [27,28]. From these fits, we have determined the van't Hoff enthalpy (ΔHvH) and the melting temperature (Tm) for Cpcyt c552 (Table 1). Since at Tm, ΔG = 0 and ΔS = ΔHvH/Tm, we can also calculate the unfolding entropy ΔS for this protein (Table 1). Limited enthalpy data are available in the literature for thermal denaturation of cyt c at neutral pH [29,30]; here we will compare to ferricytochromes c from the mesophilic eukaryotes yeast and horse and the thermophilic bacterium Thermus thermophilus.1
1 Table S1 presents a multiple sequence alignment for Cpcyt c552 with cyt c from these organisms. Although the sequence identities with Cpcyt c552 are low (15.7–20.6 %), the structures of these cytochromes are all very similar to that of the predicted structure of Cpcyt c552. Also included in the alignment is the sequence of cyt c552 from the mesophile M. hydrocarbonoclasticus, the closest match to Cpcyt c552 in the PDB.
77
For yeast iso-1-cytochrome c, a plot of ΔHvH vs. Tm is linear in the range of pH 3–5 [31,32]. Extrapolation to pH 7 yields ΔH vH = 398 kJ/mol and T m = 333 K (Table 1). It is of note that Cpcyt c 552 has a T m that is identical (within error, 334 ± 2 K) to that of yeast iso-1-cytochrome c at pH 7, while ΔH vH for Cpcyt c 552 is lower by ~ 90 kJ/mol than that of yeast iso-1-cytochrome c (ΔHvH = 309 ± 33 kJ/mol). In the case of horse heart cytochrome c, both Tm and ΔHvH are high [33]. By contrast, a thermophilic cyt c from T. thermophilus has both a higher Tm and a significantly lower ΔHvH (Table 1) [34]. One might expect that if a thermophilic cyt c is characterized by lower ΔH and ΔS and higher Tm values when compared to a mesophilic homolog, then psychrophilic proteins would be characterized by lower Tm, and increased values of ΔH and ΔS. The results reported here, however, indicate that this is not the case. Our findings reveal that psychrophilic Cpcyt c552 has a lower ΔHvH and unchanged Tm compared to similar mesophilic proteins and suggest that there exist mechanisms of protein stabilization in the psychrophile that are unexpected based on sequence alone. It will be important to examine the three-dimensional structure of the protein for insights into the molecular basis of this stabilization. In particular, it has been suggested that psychrophilic proteins have decreased intramolecular interactions (e.g., hydrogen bonds) in order to increase flexibility, with an expected concomitant decrease in stability [6,35,36]. The large number of alanines in the amino acid sequence of Cpcyt c552 (16 residues, 20.3%) is consistent with the prediction of greater protein flexibility. The relatively short, hydrophobic alanine side chain should decrease steric conflicts and increase overall protein chain flexibility, as demonstrated by the intrachain diffusion studies of Kiefhaber and coworkers [37,38]. Our work suggests that the predicted flexibility of psychrophilic Cpcyt c552 does not correlate with overall protein stability toward unfolding. Protein dynamics (flexibility) and protein stability are not simply coupled in this protein. Further calorimetric, spectroscopic, and structural studies to investigate these issues are underway. Abbreviations wavelength of maximum absorbance λmax Cpcyt c552 Colwellia psychrerythraea cytochrome c552 ΔHvH van't Hoff enthalpy IPTG isopropyl β-D-1-thiogalactopyranoside melting temperature Tm
Acknowledgments We thank Linda Thöny-Meyer for generously making the pEC86 plasmid available. We gratefully acknowledge the funding from a Camille and Henry Dreyfus Foundation Faculty Start-Up Award, a Barnard College Presidential Research Award, the National Science Foundation (MRI-R2 CHE-0959177 and ARI-R2 CHE-0961709), the New York Division of Science, Technology and Innovation (NYSTAR), the Columbia NSF NSEC: Center for Electron Transport in Molecular Nanostructures (CHE-0117752 and CHE-0641523), a grant to Barnard College from the Undergraduate Science Education Program of the Howard Hughes Medical Institute (VFO), the Barnard College Con Edison Summer Internship in Science Program (WC), the NIH Training Program in Molecular Biophysics at Columbia (PBH) (T32 GM008281), and Barnard College. No funding sources had any role in the conduct of the research or preparation of the report. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jinorgbio.2013.11.002.
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V.F. Oswald et al. / Journal of Inorganic Biochemistry 131 (2014) 76–78
Fig. 2. Thermal denaturation of ferricytochrome c552 from Colwellia psychrerythraea (sodium phosphate buffer, pH 7.0), monitored by UV–vis absorption spectroscopy.
Table 1 Thermodynamic parameters for ferricytochrome c unfolding. Protein
ΔHvH (kJ/mol)
Tm (K)
ΔS (kJ/mol K)
Reference
C. psychrerythraea cyt c552a Yeast iso-1-cyt cb Horse heart cyt cc T. thermophilus cyt c552d
309 ± 33 398 410 130
334 ± 2 333 357 389
0.925 1.20 1.15 0.334
This study [31,32] [33] [34]
a b c d
Determined spectroscopically; Soret band. Data reported ±95% confidence level. Determined spectroscopically; Soret band. Extrapolated from low pH data. Determined by differential scanning calorimetry. Determined spectroscopically; methionine ligation.
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