lysozyme and its crystalline polymorphs: the effects of

Lysozyme and its Crystalline Polymorphs: the Effects of different substrates on Lysozyme

Crystallization By Dean Sage and Shane Matthews

Written by Dean Sage

Young Scholars Program 2011

Sponsored by: Dr. Thayumanasamy Somasundaram

Abstract: Most simply, proteins are linear strands of amino acids that glob

together in a specific form, but more importantly they all perform a specific

function. Many, but far from all protein structures and even fewer functions

are currently known. Aside from pure science, pharmaceutical companies

have great use for knowing protein structure and function so they can be

modified to help treat disorders and create more efficient drugs. The

question soon becomes how, and the answer is X- Ray crystallography. In

order to do this, however, the proteins must be crystallized: this project is a

study on the polymorphs a single protein can form when in the presence of

different ions. Additionally, the different crystals were then X- Rayed to

create a diffraction pattern that can later be used to create an electron density

model of the protein, which is crucial to coming up with the specific

structure.

Introduction:

Lysozyme is a simple protein found in saliva and tears, but most

commonly extracted from hen egg whites. It was discovered in 1922 by

Alexander Flemming, and through X- Ray diffraction its structure was

uncovered in 1965 by David Chilton Phillips. The protein protects the body

by damaging bacterial cell walls (via a system that allows it to hydrolyze

vital linkages in the cell wall). Chosen because it is readily available from

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chicken (Gallus gallus domesticus) eggs, lysozyme also tends to produce

high resolution diffractions and is relatively easy to crystallize, which was

ideal for a first time experiment.

In order to help determine the structure of lysozyme, three biophysical

tests were performed (circular dichroism, UV- visual spectrophotometer, and

fluorospectrophotometer) prior to actually crystallizing the protein to

confirm the source of lysozyme was actually uncontaminated lysozyme.

Finally the crystals grown in the presence of different ions were X- Rayed

and their respective diffractions processed and analyzed to determine the

crystals’ space groups, which, given more time, could have been used to

create a three dimensional model of lysozyme.

Procedure:

To begin with, 500mL of 0.1M sodium acetate buffer was created, and

with acetic acid the pH was brought from 8 to 4.8- this will simply be

referred to as the buffer. In addition, the buffer was used to create 50mL

each of 10% (w/v) Sodium Chloride, Sodium Iodide, Sodium Nitrate, and

Potassium Thiocyanate solutions by adding in amounts respective to the

compunds’ formula weights. The final set of solutions were lysozyme in

buffer at various concentrations (45, 40, 30, 20, 15, 10)mg/mL, made one

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mL at a time in microfuge tubes. Another sample was created with

potassium nitrate at a very low concentration for the biophysical tests.

Three trays were set up in total using the hanging drop method, which

entails a drop of 2 μL lysozyme solution + 2 μL buffer suspended over 600

μL well solution (10% of the indicated compound dissolved in buffer).

45mg/mL lysozyme + NaCl



45mg/mL lysozyme + NaI

































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40mg/mL lysozyme +NaNO3

40mg/mL lysozyme + NaNO3


40mg/mL lysozyme +KSCN

40mg/mL lysozyme + KSCN














While the crystals were forming, the biophysical tests were carried out

on a single, low concentration of lysozyme in buffer. First the Circular

Dichroism, followed by spectrophotometer UV- visual, and lastly the

fluorospectrophotometer.

The next step was to observe the new crystals under a microscope and

select the best candidates for diffraction:

Good crystal: all in one piece

Needle crystal: too fragile to harvest for diffraction, which renders it useless.

In order to diffract the crystals, they had to be removed from the tray

and prepared. For cryo temperature samples, this was done with a wire loop.

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The cover slip over the well was removed, and under a microscope, a loop

used to pick up the crystal along with a thin film of buffer. The newly

suspended crystal is then inserted in the X- Ray machine

while the liquid nitrogen stream is interrupted by a brass

plate. Once the loop is secure, the brass plate is removed

as quickly as possible and the 100K stream of nitrogen flash freezes the

crystal and solution to prevent the ice interference

An unfrozen crystal mounted in a loop

that would accompany slower freezing.

The cryo samples yielded less than desirable diffractions, so an

attempt was made at 20 degrees Celsius. Because freezing isn’t an option, a

loop would quickly dry and render the crystal either lost or useless. To solve

this problem the cover slip

containing the crystal was again

removed from the well, placed

under a microscope, and instead

of being scooped up with the

loop, a glass capillary was used to trap the crystal. Once it held the crystal

and some buffer, the capillary was broken off with forceps and sealed with

capillary wax on both ends. Mounted in modeling clay, the capillary was

inserted into the X- Ray machine and diffracted.

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Regardless of temperature or suspension method within the machine,

the actual X- Raying process was very much the same. Given the negative

effects of being exposed to X – rays, everything after locking the sample in

the machine was performed remotely from a section of the lab protected by

glass infused with lead to block any scattered X rays. The computers open

the shutters, bombarding the sample with X- Rays. Behind the sample is a

digital detector, which translates the intensity picked up onto the computer

screen, thus converting X- Rays into visible light. The angles of diffraction

reveal the inner structure of the crystal.

Results:

The first usable results were the observations of the crystals, which at first

glance, revealed different structures, implying different space groups from

the same protein only due to the

different ions in solution.

Although not all of the

diffractions turned out great, some

were more than usable:

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Through computer analysis this diffraction confirmed the P 4 3 1 symmetry

of this crystal that was previously hypothesized, the other diffractions

provided conclusive evidence that P1 and P1 21 1 symmetry had been

achieved as well.

Acknowledgements:

We would like to thank FSU and all those who helped make it

possible, through the Young Scholars Program, for us to study in an

esteemed research facility. Also to Claudius Mundoma for guiding us

through the Physical Biochemistry Facility. Special thanks to our sponsor,

Thayumanasamy Somasundaram, for not only putting up with us for an

entire six weeks but showing us through the whole process of X- ray

crystallography and what really goes into making scientific research happen.

References:

"Lysozyme." Lysozyme. 2006. Web. 14 July 2011. <http://lysozyme.co.uk/>. PDB. "RCSB PDB." Protein Data Bank. RCSB. Web. 14 July 2011. <http://www.pdb.org/pdb/results/results.do?outformat=>. "X-RAY Crystallography." St. Olaf College—A Private Liberal Arts

College of the Lutheran Church in Minnesota. St. Olaf College. Web. 14

July 2011. <http://www.stolaf.edu/people/hansonr/mo/x-ray.html>.

Shane Matthews Space Groups of Lysozyme

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XRay Crystallography: The Effect of Different Substrat ozyme

es on the Crystalline Structure of Lys

by S ge

hane Matthews and Dean Sa

Written by Shane Matthews

FSU Young Scholars Program, Summer 2011

Sponsor: Dr. Thayumanasamy Somasundaram


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Abstract

X‐ray crystallography is a science that can be used to solve the

structure of any molecule that will crystallize. After analyzing the

diffraction pattern that results from diffracting a crystal, a

crystallographer can determine the electron density of the molecule,

and by extension, the structure of the molecule.

Lysozyme, an enzyme found in chicken egg whites as well as

various human secretions, is a protein which readily crystallizes and

diffracts. Lysozyme has various crystalline arrangements, known as

space groups, which can be achieved through the use of differing

substrates during the crystallization process. A space group is an

expression of the symmetry within the unit cell of the crystal. The unit

cell is the smallest repeating structure inside the crystal.

The property of having more than one possible crystalline

arrangement is known as polymorphism. When the differing space

groups are achieved through solvation, this quality is known as

pseudopolymorphism. Lysozyme crystals were grown using the hanging

drop method.

Shane MSpace Groups of L

Confirmation of differing crystalline arrangements can be

achieved through x‐ray diffraction. Analysis of the diffraction pattern

will indicate the space group of the diffracted crystal.

atthews ysozyme

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Introduction

Since the early 1950s, X‐Ray Crystallography has been the chief

technique used in solving the structure of atoms and molecules.

Crystallographers can solve the structure of any molecule that will form

a crystal, and because numerous organic, inorganic, and biological

molecules will crystallize in the right condition. After beaming

concentrated x‐rays through the crystal, a diffraction pattern is

produced. After extensive analysis of dozens of diffraction pattern

produced from the crystal at various angles, the electron density of the

molecule in question can be determined, and from this, the structure of

the molecule.

X‐ray crystallography has been used extensively to solve the

structures of thousands of proteins, as well as other molecules. Because

the structure of a protein and the function of a protein are closely

related, solving the structure of a protein or other biological molecule

can be useful in the fields of pharmaceuticals, medicine, biology, and

Shane MatthewSpace Groups of Lysozym

chemistry. Knowing the structure and the function of a protein makes

manipulation of the protein possible. Understanding a protein can help

produce new drugs in order to treat diseases. Understanding the

structure and function of molecules in general helps further scientific

esearch and development.

s e

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r

Procedure and Methods

Three trays of crystals were prepared using the hanging drop

method, with 600 mL of 10% w/v sodium acetate buffer. Several μL of

protein solution is

The substrates used during preparation of the first and second

trays were sodium chloride and sodium iodide. Although the substrates

used for the first and second crystal trays were identical, each tray had

several different concentratio sozyns of ly me.

Tray 1 Tray 2 Tray 3 Solutes NaCl, NaI NaCl, NaI NaNO3, KSCN Lysozyme concentrations (mg/mL)

45, 30, 15 40, 20, 10 40, 20, 10

The crystals were diffracted using both the loop method and the

capillary method.

Shane MatthewsSpace Groups of Lysozyme

In the loop method, a crystal is plucked from the glass slide, flash

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frozen at 100 Kelvin, and then diffracted.

In the capillary method, a crystal is manually sucked into a glass

or quartz capillary. The capillary is then sealed with wax on either side.

The benefit to this method is that liquid nitrogen is not necessary;

however, the capillary itself does produce interference during

diffraction.

Results

In terms of crystal quality, the second and third trays were much

more successful than the first, which produced only spiny crystals,

which cannot be diffracted with any meaningful results.

However, the second and third trays both produced crystals

hich were diffracted successfully. w


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Lysozyme crystals achieved in the second and third crystal trays.

Substrates used in the crystallization process, clockwise starting from

the top right: Potassium thiocyanate, sodium iodide, sodium chloride,

nd sodium nitrate. a


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These crystals were diffracted with some success.

Substrates used to crystallize the crystal that produced each respective

diffraction, clockwise starting from the top right: Potassium thiocyanate,

sodium iodide, sodium chloride, and sodium nitrate. The best diffraction

Shane MattheSpace Groups of Lysozy

was produced by the crystal crystallized using NaCl; the resolution of

the diffraction is the highest.

ws me

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Cys g d trates talline arrangements achieved usin ifferent subs

Substrate used during crystallization Space Group NaCl P4 32 12 NaI P1 NaNO3 P1 KSCN P1 21 1

Using four different substrates, three unique space groups of

lysozyme were achieved; this is a perfect example of

pseudopolymorphism.

Conclusion

The protein lysozyme crystallized successfully using different

substrates, including sodium chloride, sodium iodide, sodium nitrate,

and potassium thiocyanate. The use of different substrates during the

crystallization process of lysozyme produced crystals of varying space

groups.

X‐ray crystallography is a very valuable science, in that it has

solved the structure of thousands of molecules. Because the structure of

a molecule and function of a molecule are related, knowledge of the


structure is very valuable in that it can be used to manipulate molecules

for use in pharmaceuticals.

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lysozyme and its crystalline polymorphs: the effects of

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