A battery of micro techniques were developed which allow the screening of a large number of conditions using only a small amount of the macromolecule. The need to develop methodologies for growing large crystals required for neutron diffraction studies is discussed

Meehan, E. J., Jr.

English

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32070
CRYSTALLIZATION OF BIOLOGICAL MACROMOLECULES
IN A REDUCED GRAVITY ENVIRONMENT
by
Edward J. Meehan, Jr.^
Application of the techniques of x-ray diffraction to crystal structures
of biological macromolecules has been overwhelmingly successful. The three-
dimensional molecular structure of over 70 proteins and the nucleic acid t-RNAp e
have been determined (i). These structural studies, together with other bio-
chemical techniques, have provided a molecular basis for understanding the bio-
logical activities of enzymes, hormones, antibodies, redox, and transport pro-
teins.
There are, however, limitations associated with the use of x-ray dif-
fraction studies that can be overcome using neutron radiation. The x-ray
scattering factor of an atom is proportional to the number of electrons it con-
tains, thus scatter from hydrogen is very weak. As a consequence, the positions
of hydrogen atoms in macromolecules cannot be determined experimentally using
x-ray radiation and can only be inferred from the locations of non-hydrogen
atoms. Experimentally determined hydrogen positions would be valuable con-
sidering the importance of hydrogen bonding in maintaining protein and nucleic
acid structures, and the putative roles of hydorgen atoms in the catalytic
mechanisms pf a number of enzymes. Neutrons, on the other hand, in non-magnetic
materials are scattered by the nucleus and all atoms including light ones have
significant scattering factors. Thus, the positions of hydrogen atoms in bib-
logical macromolecules could be determined using neutron diffraction techniques.
^'Chemistry Department, The University of Alabama in Huntsville, Huntsville,
Alabama 35807
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There are other advantages offered by neutron radiation. Since it is
non-ionizing, there is no radiation damage and the entire data set may be
collected from a single crystal. X-ray induced radiation damage often necessi-
tates the use of several crystals and data from individual crystals must be
scaled together to obtain a complete data set. Absorption problems are mini-
mized using neutron radiation, while useful anomalous dispersion effects are
large. X-ray scattering factors also display a large dependence on scattering
angle and fall off rapidly at high resolution, neutron scattering factors lack
this strong angular dependence. More high resolution reflections should there-
fore be observable using neutrons, theoretically yielding more accurate results.
Despite all these advantages and the existence of excellent facilities
for collecting neutron diffraction data, very few proteins have been examined
using this technique. The difficulty and indeed the rate limiting step is the
growth of single crystals of sufficient size. The neutron flux available at
suitable wavelengths is approximately 10 times less than that available for
x-rays. A high resolution x-ray study of a protein of approximately 50,000
molecular weight would require a crystal about 0.3 mm in all dimensions, while
a neutron diffraction study would require a crystal in excess of 3 mm in all
dimensions.
Methodologies need to be developed for growing large crystals required
for neutron diffraction studies. The factors controlling nucleation and growth
of simple systems such as crystals of metals and salts have received much
attention, yet very little work has been done to understand these factors in
protein systems. It is also difficult to extend the theoretical work obtained
in these simple systems to biological macromolecules. The absence of a good
theoretical.model hinders efforts to obtain improved crystals; nevertheless,
the importance of. this class of molecules warrants their detailed study.
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Current strategies for growing large protein crystals attempt to limit
the number of nucleation sites and to very, very slowly approach the point of
insufficient solvation. This is based on evidence demonstrating the correla-
tion of crystal size and quality with the rate of growth. Amorphous precipi-
tate or many small crystals are formed if the macromolecule is rapidly forced
out of solution, while slower rates of growth result in larger, more perfectly
formed crystals. The low gravity environment of space may have special advan-
tages when trying to obtain low growth rates. In the absence of gravity driven
convection and sedimentation, diffusion becomes the principle means of material
transport. The rate of transport can then be controlled by the concentration
of the diffusing substances and by the diffusion pathlength.
When attempting to crystallize a protein or nucleic acid, the crystallo-
grapher or biochemist must examine its solubility as a function of a number of
parameters. These include the concentration of macromolecule, pH, temperature,
ionic strength, choice of buffer, choice and concentration of "precipitating
agent." Special cofactors, metal ions, or reducing agents may also be required.
The task then is to search this multiparameter space for solubility
minima. The fact that proteins commonly grow in many different crystal forms
demonstrates the possibility of many local minima (2,3). A battery of micro
techniques have been developed which allow the screening of a large number of
conditions using only a small amount of the macromolecule. Experience indicates
that if a water soluble globular protein with molecular weight less than 100,000
can be isolated in sufficient quantity and purity, the chance of obtaining
crystals are excellent. Once conditions that produce crystals have been deter-
mined, the task then is to grow them to sufficient size. Again, either micro
or macro techniques can be used, and a wide variety of methods is available.
These methods include diffusion techniques that could be readily adapted for
flight experiments.
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REFERENCES
(1) T, L. Blundell and L. N. Johnson, Protein Crystallography, Academic Press,
New York, N. Y. (1976).
(2} R. Czok and T. Bucher, Av. in Prot. Chem. 1£, 315 (1960).
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Edsall, eds., Reinhold, N. Y. (1950).
(4) H. P. Avey, R. 0. Poljak, G. Rossi and A. Nisonoff, Nature 22£, 1248 (1968),
(5) A. McPherson and A. Rich, Arch. Biochem. Biophys. 157, 23 (1973).
(6) A. Jack, J. Weinzierl and A. J. Kalb, J. Mol. Biol. 58, 389 (1971).
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(8) S. Higashi and T. Ooi, J. Mai. Biol. 34, 699 (1968).
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(10) J. Drenth and J. D. G, Smit, Biophys. Biochem. Res. Conn. 45_, 1320 (1971).
(11) C. H. Wei, J. Biol. Chem. 248, 3745 (1973). .
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Crystallization of biological macromolecules in a reduced gravity environment

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