Growth
of 3D Protein Crystals. Insulin. |
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Large, well-ordered 3D protein crystals are required for solving protein structure by X-ray
crystallography. Formation of such crystals remains the botteleneck of crystallographic studies, even for soluble proteins.
Understanding the mechanisms of crystal growth and defect formation will lead to rational
approach to crystallization and an improvement in the success rate of crystallization trials.
Proteins are excellent model systems
with which fundamental physical processes of self-assembly can be
studied.
In
vivo, proteins perform multitudes of functions.
Crystalline proteins may be useful for biotechnological purpouses.
They (the crysals) are
beautiful.
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Even
larger crystals (milimeters in size)
are required for neutron scattering studies, which are aimed at the
elucidation of the locations of hydrogen atoms in the crystals (recall
that hydrogens are invisible to X-rays and their locations are inferred
from the X-ray difraction studies). Take a look for yourself here.
To grow larger crystals, exquisit control over the growth conditions is
required. The
ability to control the growth of crystals depends on the understanding
of the various factors that affect the
crystal growth.
Crystals of proteins are already used in pharmaceudical industry. Insulin preparations used to
treat diabetis are suspensions of crystals.
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Below the roughening transition, 3D crystals
grow by spreading
of layers.
Molecules attach to the edges of these
layers (called steps) at specific sites. These attachement sites are called kinks.
Layers can be generated at screw
dislocations (see below), by two-dimensional nucleation,
or by three-dimensional
nucleation. Here
is an excellent web page by James De Yoreo from Lawrence
Livermore National Laboratory with
some atomic force microscopy images of these
processes.
A sequence of steps, such as one shown on the right, is called "step train".
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An optical micrograph of a rhombohedral (R3)
insulin 3D crystal is shown on the left. The corner indicated with solid arrows is closest
to the observer. The corner indicated with dashed arrows is furthest from
the observer. The green line connecting the two corners is drawn to
indicated one of the three-fold symmetry axes. The area imaged by AFM
is outlined in white (not to scale). The edge of the crystal is about
200 um long.
You can learn about crystal symmetries and crystal habit here,
here,
and here.
Specific information pretaining to the rhombohedral lattice can also be
found here.
The pdb code
of this
form of insulin is 4INS.
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These crystals grow by spreading
of layers generated at screw
dislocations.
The AFM image on the left shows a group of screw
dislocations, pointed to by arrowheads, on the <100> face
of a rhombohedral insulin crystal (an optical microscopy image of such a crystal is shown
above).
The image is 9.5
x 9.5 um2, Z-scale
(black-to-white) is 30 nm.
Dislocations
act as sources of steps, giving the whole
face a staircase-like appearence (especially clear along the black
line. See also the image below). These steps
are one molecule high. New
molecules join the crystal at the edges of the steps. Thus the crystal grows by spreading
of
the existing layers and generation of new ones, at the screw
dislocations. This is one of several possible modes of growth of 3D
crystals.
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Height
profile of the image
above, measured along the black line. The total height is ~ 30 nm,
while the height of the individual steps is 3.4 nm - corresponding to
thickness of one molecule of insulin.
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A movie showing the same group of screw dislocations in action. Each dislocation generates
single-molecule steps, which propagate away from the source. A series of AFM images (9.5 x 9.5 um2) of an insulin 3D crystal growingin situ
taken at approximately 50 s intervals, were used to construct this movie. |
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Step trains in insulin
crystals look like this:
The image is 11 x 11 um2.
The width of the terraces is almost the same throughout. This is an example of an equidistant
step train.
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More often, step trains in
crystals look like this:
The image is 20 x 20 um2.
Step density is seen to vary. (In this case, the variation is caused by a defect that
is located upstream (not shown)).
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The areas of high step
density seen in the image on the right are called "step bunches".
Formation of step bunches is a manifistation of instabilities that occur during step generation or growth.
It appears that equidistant step trains - step trains without bunches, such as the one shown in the left image - are
stable in the case of insulin.
This is due to the regularity of step generation at screw dislocations,
lack
of step-step interactions, and the fact that the growth
of these crystals is predominantly under transport
control.
These features make insulin crysals a very interesting model system for
studying crystal growth.
On one hand, step bunching lowers the quality and utility of the
material. On the other hand, patterns of step density may be useful for
applications in separation methods or as nano-templates.
See Gliko, Reviakine and
Vekilov (2003), PRL 90,
225503-1 - 225503-4 for detailed discussion of these
issues.
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The
core of a screw dislocation is strained. Strained areas dissolve at higher
supersaturations than non-strained areas. Image on the right shows a hollow core of a screw dislocation.
The concentration of insulin was chosen in such a way that the solution was still
supersaturated with respect to the non-strained areas (step edges) - i.e., the crystal was still
growing - but undersaturated with resepct to the strained areas, such as cores
of screw dislocations. They dissolved, leaving behind hollow channels. One such channel is visible in the center of the image (green arrowheads). Turquoise arrowheads point to insulin hexamers
that make up the crystal, while the black arrowhead in the lower left points to the step that winds around the disclocation core. Notice that the channel is not
isotropic. The anisotropy of the hollow core is the
manifistation of the anisotropy of the line tension of the steps with
respect to their crystallographic orientation (steps with different
orientations have different line tensions).
The image on the right was obtained in contact mode. |
125 nm2
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30 um2
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When the concentration of
insulin in solution is lowered to below that needed to sustain crystal growth (the
solution is said to become undersaturated), the crystal begins to
dissolve. Etch pits (image on the left) form in the place of the dislocations as they unwind.
The image on the left was obtained in tapping mode.
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The AFM studies shown here
build on the body of knowledge developed by several groups over the past years. You may be interetsted in looking up the work of prof. A. McPherson and colleagues
at UC Irvine, James
De Yoreo and colleagues from Lawrence
Livermore National Laboratory, as well as other papers on crystal growth by prof. Vekilov.
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