Journal of the Japanese Association for Crystal Growth Special Issue: The Memory of The Late Professor Ichiro Sunagawa Vol . 41 , No. 1 (2014)

18 – 18 – 日本結晶成長学会誌 Vol. 41, No. 1, 2014

Thermodynamics of Protein Crystallization: a Dialogue

with Professor Sunagawa

Peter G. Vekilov

I first met Professor Sunagawa in 1995 at the Sixth International Conference on Crystallization of Biological Macromolecules in Hiroshima. I had known of him and followed closely his papers since my time in graduate school: he was a dear friend of my advisor, Alex Chernov, and his group, Katsuo Tsukamoto and others, was working on a problem similar to mine, the mechanisms of crystallization from solution, and even using the same method, laser Michelson interferometry1,2).

By 1995, many researchers working on solution growth were intrigued by the emerging field of protein

crystallization. Protein molecules were larger than the small-molecule organic and inorganic compounds that were studied at that time and had significantly slower diffusion. This suggested an enticing opportunity to monitor the processes of their incorporation into crystals at the single molecule level; indeed, this was realized within a few years3-5). A more immediate issue facing protein crystallographers with acute physicochemical eye was that the temperature dependence of the solubility of many proteins was retrograde, i.e., increasing at lower temperature, and not “normal”, i.e., increasing at higher temperatures as

Department of Chemical and Biomolecular Engineering, and Department of Chemistry, University of Houston, Houston, TX 77204, USA E-mail: vekilov@uh.edu

Top: The author, at left, with Professors Alex Chernov and Ichiro Sunagawa at the 13th International Conference onCrystal Growth in Kyoto in 2001. Bottom: Temperature dependencies of the solubilities of the proteins hemoglobin Cand lysozyme that illustrate retrograde and normal trends, respectively. The corresponding values of the crystallizationenthalpy, ΔHcryst are shown. Hemoglobin data from Ref. 6; lysozyme data, from Refs. 8, 9.

Peter G. Vekilov: Thermodynamics of Protein Crystallization: a Dialogue with Professor Sunagawa

日本結晶成長学会誌 Vol. 41, No. 1, 2014 – 19 – 19

is typical for many small-molecule substances. Fig. 1 presents an example of retrograde solubility dependence of hemoglobin C6), a mutant that forms crystals in the red blood cells of patients with homozygous CC disease7), and of normal solubility of lysozyme8,9), a robust and easily available protein that has become the workhorse of protein biophysics.

During our first conversation, Professor Sunagawa pointed out that there is nothing special about retrograde solubility dependencies and the term normal solubility is misleading. Retrograde solubilities are known for ionic compounds, for instance cerium sulfate, and even common rock salt has a solubility which is practically independent of temperature. The temperature dependence of the solubility reflects the crystallization enthalpy: negative enthalpy corresponds to normal solubility, while positive, to retrograde. With this, Professor Sunagawa highlighted the importance and utility of fundamental thermodynamics laws for the understanding of seemingly novel behaviors in systems as complex as proteins solutions.

In later years, my group dedicated considerable effort to the study of protein solution thermodynamics. We realized that the temperature dependence of the solubility, via the van’t Hoff relation, yields a more accurate value of the crystallization enthalpy than calorimetry: the latter method may be biased by solution trapped between agglomerated crystallites that contributes to the heat effect but is not accounted in the mass balance. The first puzzle that we tackled was posed by the occurrence of positive crystallization enthalpy, which, by the second law of thermodynamics, corresponds to positive crystallization entropy. How could the association of molecules to a crystal, whereby they lose translational and rotational freedom, lead to positive entropy? We realized that the negative entropy of protein association is overpowered by the contribution of the solvent molecules that are bound to the proteins in the solution and set free as the crystallization contacts are established10.

This finding led to the realization that the solvent statured protein molecules in plays a crucial role in all classes of phenomena: binging, aggregation, phase separation, crystallization, and others11-13). More recently, it was shown that optimizations of the protein molecular surfaces leading to enhanced crystallizability in many cases work by increasing the solvent entropy contribution to the crystallization free energy14,15).

In another line of study we demonstrated that the solvent shell around protein molecules not only plays a decisive role for crystallization thermodynamics, but

also governs the protein crystallization kinetics: the free energy barrier for crystallization is largely due to the need to shed this shell at the locations of crystal contacts13,16,17). We showed that this applies not only to protein, but also to small molecule crystals, and interestingly, to the formation of fibers of sickle cell hemoglobin, the process that underlies the deadly sickle cell anemia18,19).

I met Professor Sunagawa many times in the following years. We always had engaging discussions and I was amazed at the depth of his knowledge in science and culture and the sharpness of his observations. We were together for a last time in 2009 at a symposium on interface mineralogy in Sendai. I presented the results of a joint investigation with Mihoko Maruyama, at that time a student of Katsuo Tsukamoto and an academic granddaughter of Sunagawa-sensei. Mihoko is now a professor in her own right, extending the proud dynasty of scientists following in the footsteps of this great man.

References 1. K. Maiwa, K. Tsukamoto, and I. Sunagawa: J.

Crystal Growth, 102 (1990) 43-53 2. P. G. Vekilov, Y. G. Kuznetsov, and A. A. Chernov:

J. Crystal Growth, 121 (1992) 643-655 3. S.-T. Yau, B. R. Thomas, and P. G. Vekilov: Phys.

Rev. Lett., 85 (2000) 353-356 4. D. K. Georgiou and P. G. Vekilov: Proc. Natl. Acad.

Sci. USA, 103 (2006) 1681-1686 5. G. Sazaki, M. Okada, T. Matsui, T. Watanabe,

H. Higuchi, K. Tsukamoto, and K. Nakajima: Crystal Growth & Design, 8 (6) (2008) 2024-2031

6. P. G. Vekilov, A. R. Feeling-Taylor, D. N. Petsev, O. Galkin, R. L. Nagel, and R. E. Hirsch: Biophys. J., 83 (2002) 1147-1156

7. J. E. Canterino, O. Galkin, P. G. Vekilov, and R. E. Hirsch, Biophys. J., 95 (8) (2008) 4025-4033

8. E. Cacioppo and M. L. Pusey: J. Cryst. Growth, 114 (1991) 286-292

9. S. B. Howard, P. J. Twigg, J. K. Baird, and E. J. Meehan: J. Crystal Growth, 90 (1988) 94-104

10. P. G. Vekilov, A. R. Feeling-Taylor, S.-T. Yau, and D. N. Petsev: Acta Crystallogr. Section, D 58 (2002) 1611-1616

11. P. G. Vekilov and A. A. Chernov: in Solid State Physics, edited by H. Ehrenreich and F. Spaepen(Academic Press, New York, 2002), Vol. 57, pp. 1-147

12. S. Brandon, P. Katsonis, and P. G. Vekilov: Phys. Rev. E., 73 (2006) 061917

Peter G. Vekilov: Thermodynamics of Protein Crystallization: a Dialogue with Professor Sunagawa

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13. P. G. Vekilov: Soft Matter, 6 (2010) 5254-5272 14. Z. S. Derewenda and P. G. Vekilov: Acta

Crystallogr. Section, D 62 (2006) 116-124 15. D. Fusco and P. Charbonneau: Physical Review, E

88 (1) (2013) 16. D. N. Petsev, K. Chen, O. Gliko, and P. G. Vekilov:

Proc. Natl. Acad. Sci. USA, 100 (2003) 792-796

17. P. G. Vekilov: Crystal Growth and Design, 7 (12), (2007) 2796-2810

18. P. Vekilov: Brit. J. Haematol., 139 (2), (2007) 173-184

19. P . G . V e k i l o v , O . G a l k i n , B . M . P e t t i t t , N. Choudhury, and R. L. Nagel: J Mol Biol, 377 (3), (2008) 882-888