SOLID STATE PHYSICS. VOL. 57
`
`The Physics of Protein
`Crystal I ization
`
`PETER G . VEKILOV' AND ALEXANDER A. CHERNOV~
`
`'Department of Chemical Engineering, University of Houston, Houston, TX 77546, USA
`'Universities Space Research Association, Marshall Space Flight Center; SD46
`Huntsville, AL 35812, USA
`
`1V.
`
`Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`I.
`11. Proteins and Protein Crystallization: Phenomenology . . . . . . . . . . . . . . . . . . . . . . . . . .
`1. The Protein Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2. Laboratory Techniques of Protein Crystallization
`3. Solubility Diagrams of Proteins, , , . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . , . . , . .
`111. The Protein Crystal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . . . . . . . . .
`4.
`Intermolecular Contacts
`5. Intracrystalline Solution:
`6. Elasticity and Strength.. . . . . . . . . . . . . . . . . . . . . . . . . . . .
`7. Osmotic Pressure and Cracking. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`tein Solution. . . . . . . . .
`Intermolecular Interactions and the
`....................
`8. The Colloid Stability Approach
`poferritin Solutions . . , .
`9. Interactions from Light Scatteri
`10. The Need for High Electrolyte Concentration in the Crystallization
`ofHemoglobinC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`11. Phase Diagram: Dense Liquid Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`.......................
`V. Thermodynamics and Crystallization Driving Force
`12. Interactions and Thermodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`13. Solvent Entropy Effects of the Molecular Attachment to Crystals
`14. Human Hemoglobin C-Positive Enthalpy of Crystallization . . . . . . . . . . . . . . .
`15. Apofemtin-Athermal Crystallization Driven by the Release
`......................
`of Water Molecules
`16. Lysozyme-Negative
`17. Solution Non-Ideality and the Crystallization Driving Force
`VI. Crystal Nucleation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`18. Techniques for Nucleation Rates Determinations.. . . . . . . . . . . . .
`19. Kinetics of the Nucleation Processes. . . . . . . . . . . . . . . . . . . . . . .
`20. Enhanced Crystal Nucleation Around the L-L
`2 1. Control of the Nucleation Rate of Protein Cry
`22. The Shape and Stru
`VII. Growth of Crystals: the Mesoscopic Lengthscales . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`..........................
`23. General Background
`24. Generation ofsteps ................................................
`25. Kinetics of Step Propagation, the Kinetic Coefficients
`Impurity Effects on Step Propagation . . . . . . . . . . . . .
`26.
`Interactions between Steps. . . . . . . . . . . . . . . . . . . .
`27.
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`ISBN 0-12-607757-6
`ISSN 0081-1947102 $35.00
`
`8 2002 Elsevier Science (USA)
`All rights reserved.
`
`1
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`2
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`PETER G. VEKILOV AND ALEXANDER A. CHERNOV
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`VI11. Molecular Processes at Steps . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`29. Higher-Order Neighbors and Kink Density . . . . .
`30. Molecular Potentials and Kink Density. . . . . . . . .
`
`. . . . . . . . . . . . . . . . . .
`. . . . . . . . . . . . . . . . . .
`
`X. Non-uniform Step
`XI. Crystal Perfection
`36. Sub-Molecu
`37. Rotational a
`
`...................................
`
`1 Lattice Defects
`
`. . . . . . . . . . . . . . . . . .
`
`...................................
`
`42. Mosaicity . . .
`43. Growth under
`Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`. . . .
`Acknowledgments .
`. . . . . . . . . . .
`
`. . . . . . . . . . . . . . . . . .
`
`XII.
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`104
`106
`108
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`1 1 1
`1 1 1
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`1 I3
`115
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`143
`146
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`
`1. Introduction
`
`Proteins are the elementary building units of all living creatures and essential
`components for information and energy processing within the living systems.’
`The correlation between the structure and the function for this group of natural
`compounds has been the focus of intense investigations for more than 50 years.
`(For a brief history, see Ref. 2, and for further and more recent developments see
`Refs. 3-6.) Currently, the two most broadly used methods of protein structure
`determinations are x-ray crystallography and Nuclear Magnetic Resonance
`(NMR). NMR is limited to proteins of molecular mass lower than 30,000-
`60,000 Da, involves rather long data collection time^,^-^ and thus is expected to
`
`’ T. E. Creighton, Proteins: Structure and Molecular Properries, W. H. Freeman, New York (1993).
`* A . McPherson, J. Cryst. Growth 110, 1-10 (1991).
`M. F. Perutz and F. S. Mathews, J. Mol. Biol. 21, 199-202 (1966).
`S. R. Simon, W. H. Konigsberg, W. Bolton, and M. F. Perutz, J. Mol. Biol. 28, 451454 (1967).
`A. McPherson, Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press,
`New York (1999).
`A. McPherson, A. J. Makin, andY. G. Kuznetsov, Ann. Rev. Biomol. Struct. 20, 361410 (2000).
`’ K. Wiitrich, Acra Cryst. Section D 51, 249-270 (1995).
`W. S. Warren, Science 280, 398-399 (1998).
`S. J. Glaser, T. Schulte-Herbriiggen, M. Sieveking, 0. Schedletzky, N. C. Nielsen, 0. W. S~rensen,
`and C. Griesinger, Science 280, 421424 (1998).
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`THE PHYSICS OF PROTEIN CRYSTALLIZATION
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`3
`
`contribute at most 20% of the data. On the other hand, due to recent advances
`in protein expression and isolation," data collection on specialized high-intensity
`synchrotron beam lines,"*'2 crystallization screening technique^,'^-'^ and compu-
`tational methods for structure determination and refinement, x-ray crystallography
`is close to becoming a high-throughput method."
`To resolve atoms that are, typically, 1.5-2 A apart, the diffraction methods require
`single crystals that are as large as several tenths of a millimeter in all three dimen-
`sions, and have low defect contents and high compositional and structural uniformity.
`At present, most proteins can be enticed to produce some kind of microcrystals.
`However, the crystals are often of significantly lower quality and/or size than needed
`for. the desired resolution. Often, further improvement in crystallization involves
`the application of elaborate, protein-specific techniques. A few non-exhaustive
`examples include genetic engineering of intermolecular contact^,'^'^^ proteolysis,21
`utilization of the phase behavior of complex, multicomponent system^?^.*^ or
`even costly microgravity experiments aboard
`Hence, claims that
`protein crystallization is a major rate-limiting step in structure determinations
`
`10
`
`22
`
`S. K. Burley, S. C. Almo, J. B. Bonanno, M. Capel, M. R. Chance, T. Gaasterland, D. Lin, A. Sali,
`F. W. Studier, and S. Swaminathan, Nurure Generics 23, 151-157 (1999).
`' I S.-H. Kim, Nurure Sfrucf. Biol. Synchrotron Supplement 6 4 3 4 5 (1998).
`l2 R. S. Service, Science 285, 1342-1346 (1999).
`l3 N. E. Chayen, P. D. ShawStewart, and P. Baldock, Acru Crysrullog,: Section D 50,456-458 (1994).
`l4 B. Cudney, S. Pattel, K. Weisgraber, Y. Newhouse, and A. McPherson, Acru Crystallog,: Secrion D
`50,414423 (1994).
`Is G. L. Gilliland, M. Tung, D. M. Blakeslee, and J. E. Ladner, Acru Crystallog,: Section D 50,
`408-413 (1994).
`16
`L. F. Kuyper and C. W. Carter Jr., J. Cryst. Growth 168, 155-169 (1996).
`B. D. Prater, S. C. Tuller, and L. J. Wilson, J. Cryst. Growrh 196, 67&684 (1999).
`IS
`P. D. S. Stewart and P. F. M. Baldock, J. Cryst. Growrh 196, 665-673 (1999).
`l9 D. M. Lawson, P. J. ~rtymiuk, S. J. Y e w m , J. M. A. smith, J. C. Livingstone, A. TE~~Y, A. Luzzago,
`S. Levi, P. Arosio, G. Cesareni, C. D. Thomas, W.V. Shaw, andP. M. Harrison, Nur~re349,541-544 (1991).
`20
`D. A. Doyle, J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Culbis, S. L. Cohen, B. T. Chait, and
`R. MacKinnon, Science 280,69-77 (1998).
`S . L. Cohen, A. R. Ferre-D' Amare, S. K. Burley, and B. T. Chait, Protein Sci. 4, 1088-1099 (1995).
`E. Pebay-Peyroula, G. Rummel, J. P. Rosenbusch, and E. M. Landau, Science 277,1676-1681 (1997).
`E. M. Landau and J. P. Rosenbusch, Proc. Nurl. Acud. Sci. USA 93, 14532-14535 (1996).
`L. J. DeLucas and C. E. Bugg, Trends in Biotechnology 5, 188-193 (1987).
`J. Dong, T. Boggon, N. Chayen, J. Raftery, R. Bi, and J. Helliwell, Acru Crystallog,: D B i d .
`Crystallog,: 55, 745-752 (1999).
`26
`D. C. Carter, B. S. Wright, T. Y. Miller, J. Chapman, P. D. Twig, K. Keeling, K. Moody, M. White, J.
`Click, J. Ruble, J. X. Ho, L. Adhock-Downeey, G. Bunick, and J. Harp, J. Ctysr. Growth 1%. 6024W (1999).
`27
`D. C. Carter, B. S. Wright, T. Y. Miller, J. Chapman, P. D. Tivigg, K. Keeling, K. Moody, M. White,
`J. Click, J. Ruble, J. X. Ho, L. Adhock-Downeey, T. Dowling, C.-H. Chang, P. Ala, J. Rose, B. C.
`Wang, J.-P. Declerq, C. Evrard, J. Rosenberg, J.-P. Wery, D. Clawson, M. Wardell, W. Stallings, and
`A. Stevens, J. Crysr. Gmwrh 196, 61M22 (1999).
`28
`E. H. Snell, A. Cassetta, J. R. Helliwell, T. J. Boggon, N. E. Chayen, E. Weckert, K. Hoelzer, K.
`Schroer, E. J. Gordon, and P. F. Zagalski, Acfu Crystallog,: Section D 53, 231-239 (1997).
`
`23
`
`24
`
`25
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`PETER G. VEKILOV AND ALEXANDER A. CHERNOV
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`5.29-32
`and reflect the fact that a determined effort
`abound, even in recent literature,
`that will eventually produce suitable crystals may take several years.
`Beyond protein single crystal growth, progress in various biochemical and bio-
`medical research and production tasks is impeded by lack of insight into protein
`nucleation and growth mechanisms. For instance, the slow dissolution rate of
`protein crystals is used to achieve sustained release of medications, such as insulin,
`interferon-a, or the human growth
`Work on the crystallization of
`other therapeutically active proteins+.g., antibodies for foreign proteins-so
`they
`can be dispensed in a similar manner, is currently underway. If the administered
`dose consists of a few, larger, equidimensional crystallites, steady medication
`release rates can be maintained for longer periods than for doses composFd of many
`smaller crystallites. To achieve such size distributions, crystal nucleation times must
`be short so that all crystals grow at the same decreasing supersaturation.
`Other, more rare biomedical applications relying on insight into the formation
`of protein solid phases include situations where pathological conditions are
`related to the formation of crystals or other ordered solid aggregates in the human
`body. Two often-cited examples are the crystallization of hemoglobin C and the
`polymerization of hemoglobin S, which cause, respectively, CC and sickle cell
`diseases.3w Another example is formation of a denser liquid phase in the eye
`retina, which underlies the pathology of cataract f~rmation.~’
`
`34
`
`29 P. Weber, Advances in Protein Chemisrry, Vol. 41, eds. C. B. Afinsen, F. M. Richards, J. T. Edsal,
`and D. S. Eisenberg, Academic Press, New York (1991).
`30
`A. Ducruix and R. Giege (Eds.), Crystallization of Nucleic Acids and Proteins. A Practical
`Approach, IRL Press, Oxford (1992).
`” N . E. Chayen, T. J. Boggon, A. Casseta, A. Deacon, T. Gleichmann, J. Habash, S. J. Harrop, J. R.
`Helliwell, Y. P. Neih, M. R. Peterson, J. Raftery, E. H. Snell, A. Hadener, A. C. Niemann, D. P. Siddons,
`V. Stojanoff, A. W. Thompson, T. Ursby, and M. Wulff, Quart. Rev. Biophys. 29, 227-278 (1996).
`32
`P. C. Weber, Methods in Enzymology, Vol. 276, eds. C. W. Carter Jr. and R. M. Sweet, Academic
`Press, New York (1997), 13-22.
`33
`J. Brange, Galenics of Insulin, Springer, Berlin (1987).
`M. L. Long, J. B. Bishop, T. L. Nagabhushan, P. Reichert, G. D. Smith, and L. J. DeLucas, J. Crysr.
`Growth 168, 233-243 (1996).
`” S. Matsuda, T. Senda, S. Itoh, G. Kawano, H. Mizuno, and Y. Mitsui, J. Biol. Chem. 264, 13381-
`13382 (1989).
`36
`S. Peseta, J. A. Langer, K. C. Zoon, and C. E. Samuel, Annual Review of Biochemistry, Vol. 56, eds.
`C. C. Richardson, P. D. Boyer, I. B. Dawid, and A. Meister, Annual Reviews, Palo Alto (1989). 727-778.
`37
`P. Reichert, C. McNemar, N. Nagabhushan, T. L. Nagabhushan, S. Tindal, and A. Hruza, Metal-
`Interferon-Alpha Crystals, in: 5,441,734. (US Patent, 1995).
`38 S. Charache, C. L. Conley, D. F. Waugh, R. J. Ugoretz, and J. R. Spurrell, J. Clin. Invest. 46, 1795-
`1811 (1967).
`39
`R. E. Hirsch, C. Raventos-Suarez, J. A. Olson, and R. L. Nagel, Blood 66, 775-777 (1985).
`4u
`W. A. Eaton and J. Hofrichter, Advances in Prorein Chemistry, Vol. 40, eds. C. B. Anfinsen, J. T.
`Msal, F. M. Richards, and D. S. Eisenberg, Academic Press, San Diego (1990). 63-279.
`4’ C. R. Berland, G. M. Thurston, M. Kondo, M. L. Broide, J. Pande, 0. Ogun, and G. B. Benedek, Proc.
`Narl. Acud. Sci. USA 89, 1214-1218 (1992).
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`THE PHYSICS OF PROTEIN CRYSTALLIZATION
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`Of all processes of molecular self-assembly in the biological world, the crystal-
`lization of the protein molecules is probably the best-studied example. Other exam-
`ples include the formation of quaternary protein structures, protein and protein
`nucleic acid complexes whose assembly into structural units is often essential to
`chains of coupled functions, and assembly of viri and of complex intracellular
`organelles such as the ribosome.424 Similar to crystallization, the self-assembly
`processes are enabled by molecular recognition. Hence, insight into the molecular
`mechanisms of protein crystallization could provide guidance into these related areas.
`In addition to its medical and biotechnological significance, protein crystalli-
`zation has much in common and may provide an insight into crystallization
`phenomena that occur in a variety of systems:5
`such as water freezing in clouds
`and oceans:6 magma solidification in the Earth’s interior,47 the pulling of semi-
`conductor
`and so on. Given the resolution limits of modem surface
`characterization techniques, proteins are particularly attractive for studies of
`fundamental crystal growth mechanisms. For example, the size of the protein
`molecules (a few nanometers) and the time-scales for growth (up to a few seconds
`between sequential discrete molecular attachment events) are within the reach of
`current advanced experimental techniques. On the other hand, the molecular
`masses typical of most protein molecules still leave the thermal equilibration
`times relatively short. Thus, conclusions drawn from studies of protein model
`systems may still be meaningful for small molecule crystallization. In this regard,
`proteins could be a better model than, for instance, colloidal crystal^.^^'^^
`The preceding factors led to the emergence of macromolecular crystallization
`as a distinct area of research in the early 1980s. Since then, the field has benefited
`from concepts and methods developed in other research areas. For instance, the
`application of direct light scattering and other methods used to probe colloids
`led to quantitative measurements of molecular interactions and crystal nucleation
`
`in protein s ~ l u t i o n s . ~ ’ - ~ ~ Fluid dynamics analyses were applied to characterize
`the convective4iffusive supply fields in the solutions from which the crystals
`
`42
`
`43
`
`44
`
`45
`
`46
`
`47
`
`48
`
`N. Ban, P. Nissen, J. Hansen, P. B. Moore, and T. A. Steitz, Science 289, 905-920 (2000).
`P. Nissen, J. Hansen, N. Ban, P. B. Moore, and T. A. Steitz, Science 289, 920-930 (2000).
`K. Wild, 1. Sinning, and S. Cusack, Science 294, 598401 (2001).
`F. Rosenberger, Crysr. Res. Technol. 34, 163-165 (1999).
`D. W. Oxtoby, J. Phys.: Condensed Matter 4, 7627-7650 (1992).
`0. Melnik and R. S. J. Sparks, Narure 402, 37-41 (1999).
`H.-W. Ren, X. Q. Shen, and T. Nishinaga, J. Crysr. Growth 166, 217-221 (1995).
`P. N. Pusey and W. Van Megen, Nature 320, 340-343 (1986).
`W. Van Megen and S. M. Underwood, Nature 362, 6 1 M 1 9 (1993).
`” 2. Kam and J. Hofnchter, Eiophys. J. 50, 1015-1020 (1986).
`52
`A. George and W. W. Wilson, Acta Crystallogr: Section D 50, 361-365 (1994).
`M. Muschol and F. Rosenberger, J. Chem. Phys. 103, 10424-10432 (1995).
`F. Rosenberger, P. G. Vekilov, M. Muschol, and B. R. Thomas, J. Crysr. Growth 167, 1-27 (1996).
`A. J. Malkin and A. McPherson, Acra Crystallogr: Section D 50, 385-395 (1994).
`
`49
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`50
`
`53
`
`54
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`PETER G . VEKILOV AND ALEXANDER A. CHERNOV
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`5 6 5 9
`Explanations of the differences between terrestrially grown protein crys-
`grow.
`tals and those grown in microgravity have largely been based on such analyses.
`Interferometric and scanning probe techniques, first used by inorganic crystal
`-3
`growers
`and surface scientists, provided insight into growth processes on a near-
`molecular
`Different methods to establish and maintain advantageous
`supersaturation, temperature, and solution composition conditions have helped to
`achieve improved reproducible perfection of protein crystal^.^'^"^
`A massive recent effort, the Structural Genomics initiative within the broader
`Proteomics project, is directed toward determination of the structures of proteins from
`10.7 I .7?
`a broad variety of species using x-ray crystallography.
`From the protein structure,
`the function of the respective protein can be understood on atomic level and/or deduced
`with a high degree of fidelity. The goal is to establish a statistically viable correlation
`between the now available sequence of amino acids in the protein chain (directly
`linked with the sequence of bases in the ribonucleic acids coding for the protein) and
`the special folding of this chain. This folding provides a structure of the globular
`10.71.73
`protein molecule, determines the function of the protein and the associated gene.
`The nature of the project allows for a random selection of the target proteins, and
`hence, if a protein does not succumb to crystallization within the time frame of a
`10,7677
`high-throughput methodology, it is abandoned for another protein.
`For this goal,
`
`56
`
`57
`
`5n
`
`59
`
`M. Pusey, W. Witherow, and R. Naumann, J. Cryst. Growth 90, 105-1 11 (1988).
`H. Lin, F. Rosenberger, J. I. D. Alexander, and A. Nadarajah, J. Cryst. Growth 151, 153-162 (1995).
`N. Ramachandran, C. R. Baugher, and R. J. Naumann, J. Crysr. Growth 8, 17G179 (1995).
`F. Rosenberger, J. Cryst. Growth 76,618436 (1986).
`60
`L. N. Rashkovich, A. A. Mkrtchan, and A. A. Chernov, Sov. Phys. Crystallog,: 30,350-355 (1985).
`A. A. Chernov, L. N. Rashkovich, I. L. Smol’sky, Y. G. Kuznetsov, A. A. Mkrtchan, and A. I.
`Malkin, Growth of Crysrals, Vol. 15, ed. E. E. Givargizov, Consultant Bureau, New York (1986).
`62
`A. J. Gratz, P. E. Hillner, and P. K. Hansma, Geochimica and Cosmochimica Acta 57,491495 (1993).
`P. G. Vekilov, M. Ataka, and T. Katsura, J. Cryst. Growth 130, 317-320 (1993).
`64
`P. G. Vekilov, Sfudies and Concepts in Crysral Growth, ed. H. Komatsu, Pergamon, Oxford ( 1993). 2549.
`Y. G. Kuznetov, A. J. Malkin, A. Geenwood, and A. McPherson, J. Struct. Biol. 114, 184196 (1995).
`Y. G. Kuznetsov, A. I. Malkin, and A. McPherson, J. Cryst. Growth 196,489-502 (1999).
`A. J. Makin, T. A. Land, Y. U. G. Kuznetsov, A. McPherson, and J. J. DeYoreo, Phys. Rev. Left.
`75, 2778-2781 (1995).
`68
`T. A. Land, J. J. DeYoreo, and J. D. Lee, Surf: Sci. 384, 136155 (1997).
`69
`C. M. Yip, M. R. DePhelippis, B. H. Frank, M. L. Brader, and M. D. Ward, Biophysical J. 75,
`1172-1 179 (1998).
`70
`A. McPherson, Preparation and Analysis of Protein Crystals, Wiley, New York (1982).
`7’ T. I. Zarembinski, L.-W. Hung, H. J. Mueller-Dieckmann, K. K. Kim, H. Yokota, R. Kim, and S.-H.
`Kim, Proc. Narl. Acad. Sci. USA 95, 15189-15193 (1998).
`72
`A. Sali, Nurure Sfruct. Biol. 5, 1029-1032 (1998).
`R. Sanchez and A. Sali, Proc. Natl. Acad. Sci. USA 95, (1998).
`J. U. Bowie, R. Luethy, and D. Eisenberg, Science 253, 164-166 (1991).
`R. Luethy, J. U. Bowie, and D. Eisenberg, Nature 356, 83-86 (1991).
`S. Henikoff, E. A. Greene, S. Pietrokovski, P. Bork, A. K. Atwood, and L. Hood, Science 278,
`609414 (1997).
`77
`H. Li, C. Tang, and N. S. Wingreen, Proc. Natl. Acad. Sci. USA 95, 49874990 (1998).
`
`63
`
`65
`w
`67
`
`73
`
`74
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`7
`
`high-throughput crystallization techniques have been developed that rely heavil on
`Y
`automated screening of a huge number of samples of crystallizing solutions.’””’ ’ As
`already noted, at our current level of empirical data incorporated into the screening
`procedures, about half the proteins tested yield crystals of sufficient size and perfection
`for a structure determination, enough to ensure the viability of the structural genomics
`project. In this context, it is important to emphasize that further developments of the
`science of protein crystallization are still needed, and they will serve additional
`biological and biomedical tasks. An immediate example for the inapplicability of the
`high-throughput methods is the study of a metabolic pathway consisting of several
`tens of proteins. Such an investigation requires precise structural data of all the
`member proteins, with the associated difficulties in crystallizing at least half of them.
`Transition from art to science in crystal growing of small molecules started in
`the 1950~.’~ It brought about the robust science of crystal growth, with strong
`applications in the technology of semiconductors, lasers, magnetic, optical and
`other functional crystals and constructive materials, in industrial crystallization
`of fertilizers, drugs, paints, and so on. Fast progress is still being made in this
`field. Similar developments are likely in the protein crystallization field, though
`this task of significantly higher complexity requires the joint efforts of biochemists,
`chemists, physicists, and material scientists. These efforts can build on the progress
`during the last couple of decades. The existing broad international collaboration
`in this field is reflected in the eight International Conferences on Crystallization
`of Biological Macromolecules held since 1 986.8m7
`
`~
`
`78
`
`80
`
`J. R. Luft, J. Wolfley, I. Jurisica, J. Glasgow, S. Fortier, and G. T. DeTitta, J. Cryst. Growth 232,
`591-595 (2001).
`79
`J. J. Gilman (Ed.), The Art andScience of Growing Crystals, J. Wiley & Sons, New York, London (1963).
`R. S. Feigerlson (Ed.), Proceedings of the First International Conference on Crystallization of Bio-
`logical Macromolecules, Stanford California, August 14-16.1986, J. Cryst. Growth 76,529-718 (1986).
`R. Giege, J. C. Fontecilla-Camps, R. S. Feigelson, R. Kern, and A. McPherson (Eds.), Proceedings
`of the Second International Conference on Crystallization of Biological Macromolecules, Strasbourg,
`France, July 19-25, 1987, J. Cryst. Growth 90, 1-374 (1988).
`82
`K. Ward (Ed.), Proceedings of the Third International Conference on Crystallization of Biological
`Macromolecules, Washington, DC, August 13-19, 1989, J. Cryst. Growth 110, 1-338 (1991).
`83
`J. J. Stezowski and W. Littke (Eds.), Proceedings of the Fourth International Conference on Crystal-
`lization of Biological Macromolecules, Freiburg, Germany, 1991, J. Cryst. Growrh 122, 1 4 0 5 (1992).
`84
`J. P. Glusker (Ed.), Proceedings of the Fiffh International Conference on Crystallization of Bio-
`logical Macromolecules. San Diego, CA, USA, August 8-13, 1993, Vol. 50, 337-666 (1994).
`85
`K. Miki, M. Ataka, K. Fukuyama, Y. Higuchi, and M. T. Yashita (Eds.), Proceedings of the Sixth
`International Conference on Crystallization of Biological Macromolecules, Hiroshima, Japan, November
`12-17, 1995, J. Cryst. Growth 168, 1-327 (1996).
`86
`J. Drenth and J. M. Garcia-Ruiz (Eds.), Proceedings of the Seventh International Conference on
`Crystallization of Biological Macromolecules, Granada, Spain, May 3-8, 1998, J. Crysr. Growth 196,
`185-720 (1 999).
`87
`G. DeTitta, H. Einspar, P. G. Vekilov, and W. W. Wilson (Eds.), Proceedings of the Eighth
`International Conference on Crystallization of Biological Macromolecules, San Destin, Florida, 2000,
`J. Cryst. Growth 232, (2001).
`
`MYLAN INST. EXHIBIT 1095 PAGE 7
`
`MYLAN INST. EXHIBIT 1095 PAGE 7
`
`

`

`8
`
`PETER G. VEKILOV AND ALEXANDER A. CHERNOV
`
`Most of the investigations of the physics and physical chemistry of protein
`crystallization were necessarily carried out using just a few model protein and
`virus systems: lysozyme, canavalin, the satellite tobacco mosaic virus, thaumatin,
`the fenitidapofemtin pair, hemoglobin, insulin, and a very few other proteins and
`plant viruses. From a certain point of view, this selection is quite representative-the
`studied proteins come from sources varying from bacteria to humans, have molec-
`ular weights in the range from 7,000 to more than a million, with functions ranging
`from storage proteins, through hormones, to enzymes and viri. Although none of
`these proteins is an integral membrane protein-which
`have a hydrophobic mid-
`section rendering them insoluble in water-crystallization practice shows that after
`a membrane protein has been solubilized by wrapping it in a suitable detergent
`micelle,'* its crystallization is similar to the crystallization of the globular proteins.
`This belief is supported by a recent AFM visualization of the growing faces of
`bacteriorh~dopsin.'~ A recent study of the crystallization of a messenger RNA
`revealed that even the growth of crystals of such remotely similar macromolecules
`is akin to the growth of protein crystakgO
`The similarities between the studied protein systems, and good correspon-
`dence of the observations to the data and theories accumulated over 100 years
`of crystallization research support the conclusion that the mechanisms discussed
`here underlie the crystal growth of most macromolecules. The observations
`reviewed here also, in all likelihood, adequately represent major physical fea-
`tures of the protein crystallization processes. On the other hand, these few models
`are a drop in the sea of more than 10,000 proteins that have currently been
`crystallized. Thus, they cannot cover the elaborate interplay between these pro-
`cesses for each protein system of interest. This interplay is determined by the
`size, structure, and function of the protein, by the amino acid residues exposed
`to the surface of the molecule and their distribution, by the propensity of the
`protein molecules to associate with other molecules of the same kind, other
`proteins, or nonprotein molecules-lipids, carbohydrates, small organic or inor-
`ganic species present in the solution. In each concrete case, the phenomena
`unique to the concrete protein under study, underlying its chemical and biolog-
`ical specificity, occur simultaneously with the physical phenomena discussed
`here, modify them, alter their interplay and their effects, and even completely
`mask them.
`In this chapter, we first outline the basic experimental procedures used in the
`laboratory to grow protein crystals. We start the discussion with an overview of
`
`89
`
`8R
`C. Ostermeier and H. Michel, Curr. Opin. Sfrucf. B i d . 7 , 697-701 (1997).
`A. McPherson, A. J. Malkin, Y. G. Kuznetsov, and M. Plomp, Acru Crysfullog,: D B i d . Crysfullogr.
`57, 1053-1060 (2001).
`90
`J. D. Ng, Y. G. Kuznetsov, A. J. Makin, G. Keith, R. Giege, and A. McPherson, Nucleic Acids
`Res. 25, 2582-2588 (1997).
`
`MYLAN INST. EXHIBIT 1095 PAGE 8
`
`MYLAN INST. EXHIBIT 1095 PAGE 8
`
`

`

`THE PHYSICS OF PROTEIN CRYSTALLIZATION
`
`9
`
`the general features of crystals of biological macromolecules. Then we look into
`the interactions between the protein molecules in solution that underlie the ther-
`modynamics of the phase transitions, and at the resulting phase diagram of the
`protein solution. The discussion of crystallization proper begins, of course, with
`nucleation, followed by the growth processes on the mesoscopic and the molec-
`ular length scales. We then look at the behavior of sequences of steps and the
`arising dynamics powered by the interactions between these steps. We close with
`an overview of the mechanisms of defect formation, and the means to minimize
`the defects and enhance the crystal perfection.
`
`II. Proteins and Protein Crystallization: Phenomenology
`
`1. THE PROTEIN MOLECULES
`
`The protein molecules are linear polymers of a-amino acids. These amino acids
`are defined by the side chain, R, attached to the a-amino acid moiety, H,N-C,HR-
`COOH. Table 1 lists the twenty amino acids, which comprise all naturally occur-
`ring proteins, and provides some of their properties needed for the further dis-
`cussion. The molecular weight of an amino acid is -1 10-130 g mol-'. Among
`the twenty amino acids, three are chemically basic, two are acidic, seven are
`polar. The other eight are nonpolar and, thus, hydrophobic. In the folded protein,
`the hydrophilic acidic, basic, or polar side chains are mainly exposed to the
`aqueous solution.'
`The length of the protein polypeptide chains varies from less than a hundred
`to several thousand amino acids, resulting in molecular weights in the range of
`104-106 g mol-'. The amino acid sequence determines the way the chain is folded
`into globulae in the aqueous environment of the cytosol. A globula may include
`several folded chains. All protein functions are determined by the spatial arrange-
`ment of the side chains R.9' Hence, the three-dimensional protein structure is of
`major interest to various fields of biology.'
`
`2. LABORATORY TECHNIQUES OF PROTEIN CRYSTALLIZATION
`
`Expression, extraction, and purification of a protein are often time-consuming
`procedures. Hence, wide use is being made of various micro- methods of protein
`crystallization, employing microdialysis and microdiffusion cells, allowing one
`
`91 A. Fersht, Structure and Mechanism in Protein Science, W. H. Freeman, New York (1999).
`
`MYLAN INST. EXHIBIT 1095 PAGE 9
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`
`

`

`
`
`TABLE 1. AMINO ACIDS: S T R U ~ R E FEATURES,
`
`(FOR DETAILS, SEE REF. 107)
`IONIZATION CONSTANTS, AND RELATIONS TO THE AQUEOUS ENVIRONMENT
`
`Names
`Abbreviations,
`MW
`
`Aspartic acid
`Asp (D), 133
`Glutamic acid
`Glu (E), 147
`Arginine
`Arg (R), 174
`Histidine
`His (H), 155
`
`Lysine
`Lys (K), 146
`Asparagine
`Asn(n), 132
`Cysteine
`c y s o , 121
`Glutamine
`Gln(Q), 146
`Serine
`Ser(S), 105
`Threonine
`Thr(T), 119
`Tryptophan
`Trp(W), 204
`
`Structure and Ionization pK of Side Chains,
`Carboxyl, and Amino Groups
`PK
`C0,H; NH,
`
`Side Chain
`
`pK
`
`PI
`
`2.98
`
`3.08
`
`2.10; 9.82
`
`2.10; 9.47
`
`3.86
`0
`H-OCCH,-A~'
`4.07
`0
`H-OC(CH,),-A
`12.48 NH,
`H' HNCNH(CH,),-A
`6.10
`HC+N
`
`CH2-A
`
`10.53
`H'+H,N( CH,).,-A
`(0.3)b' 0
`H++H,NCCH,-A
`8.35
`
`H-SCHZ-A
`(0.3)b' 0
`H++H,NC(CHJ~-A
`( 16)''
`
`H-OCH,-A
`(16)L' CH,
`H-OCH-A
`
`2.01; 9.04
`
`10.76
`
`1.77; 9.18
`
`7.64
`
`2.18; 8.95
`
`9.74
`
`2.02; 8.80
`
`5.41
`
`1.86; 10.34
`
`5.11
`
`2.17; 9.13
`
`2.21; 9.15
`
`5.70
`
`5.68
`
`2.09; 9.10
`
`5.60
`
`N
`H
`
`2.38; 9.39
`CHZ-A
`
`5.88
`
`Probability
`of Residence
`on Surface
`
`Hydrophobicity
`Relative to Glycine
`(exp.)
`
`Side Chain
`Hydration
`Potential (calc.)
`
`Number in
`Lysozyme
`Molecule
`
`0.86
`
`0.80
`
`1 .oo
`
`0.81
`
`0.96
`
`0.90
`
`0.53
`
`0.94
`
`0.76
`
`0.75
`
`0.77
`
`-0.77
`
`-0.64
`
`-1.01
`
`0.13
`
`-0.99
`
`-0.6
`
`1.54
`
`-0.22
`
`-0.004
`
`0.26
`
`2.25
`
`-10.95
`
`-10.20
`
`-19.92
`
`-10.27
`
`-9.52
`
`-9.68
`
`-1.24
`
`-9.38
`
`-5.06
`
`-4.88
`
`-5.88
`
`7
`
`2
`
`11
`
`1
`
`6
`
`14
`
`8
`
`3
`
`10
`
`7
`
`6
`
`-
`
`0
`
`3
`;a
`n
`s
`F
`9
`%
`;
`E
`
`P
`?
`0
`
`B
`
`9
`
`MYLAN INST. EXHIBIT 1095 PAGE 10
`
`MYLAN INST. EXHIBIT 1095 PAGE 10
`
`

`

`Tyrosine
`Qr(Y), 181
`Alanine
`Ala(A), 89
`G 1 y c i n e
`Gly(G), 75
`Isoleucine
`Ile(I), 131
`Leucine
`Leu(L), 131
`Methionine
`Met(M), 149
`Phenylalanine
`Phe(F), 165
`Proline
`Pro(P), 115
`Valine
`Val(V), 117
`
`H - 0
`
`10.07
`
`CH,-A
`
`H-A
`
`H3C
`CH,CH,CH- A
`
`(CH&CHCH,-A
`
`CH,S(CH&-A
`
`CHC0,H
`
`(CH,),CH- A
`
`CH2-A
`NH
`
`2.20; 9.1