`
`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.
`
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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 .
`. . . . . . . . . . .
`
`. . . . . . . . . . . . . . . . . .
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`XII.
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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).
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`THE PHYSICS OF PROTEIN CRYSTALLIZATION
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`3
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`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
`
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`S. K. Burley, S. C. Almo, J. B. Bonanno, M. Capel, M. R. Chance, T. Gaasterland, D. Lin, A. Sali,
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`D. C. Carter, B. S. Wright, T. Y. Miller, J. Chapman, P. D. Twig, K. Keeling, K. Moody, M. White, J.
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`E. H. Snell, A. Cassetta, J. R. Helliwell, T. J. Boggon, N. E. Chayen, E. Weckert, K. Hoelzer, K.
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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
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`A. Ducruix and R. Giege (Eds.), Crystallization of Nucleic Acids and Proteins. A Practical
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`P. C. Weber, Methods in Enzymology, Vol. 276, eds. C. W. Carter Jr. and R. M. Sweet, Academic
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`J. Brange, Galenics of Insulin, Springer, Berlin (1987).
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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
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`43
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`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,
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`THE PHYSICS OF PROTEIN CRYSTALLIZATION
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`
`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
`
`MYLAN INST. EXHIBIT 1095 PAGE 9
`
`
`
`
`
`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