Lysozyme Crystals Lab Report Chem 351

7/23/2019 Lysozyme Crystals Lab Report Chem 351

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Lysozyme Crystals 1

Lysozyme Crystals

Growth of Lysozyme Crystals

Abstract

For x-ray crystallography, ideal protein crystals are needed in order to get viable results

for the protein structure of protein molecules. The best crystals can be made by optimizing theconditions for making the best lysozyme crystals. This is done by using different precipitant

agents along with different concentrations of the precipitant concentrations and protein

concentrations. The best salt solutions found was NaCl and NaBr. This was discovered by using

different multiple wells with different conditions. The results were compiled from multiple

sources and compared.

Introduction

In order to determine the structure of the protein molecules by x-ray crystallography, it is

essential to grow crystals of the protein. By utilizing systematic trials in order to use optimal

growth conditions, the changes of growing good crystals goes up. One widely used technique is

the hanging-drop vapor-diffusion method. In this lab, lysozyme crystals were grown using this

technique. This is due to the exchange of water vapor between the drop and the reservoir. This

will result in the drop shrinking and leaves crystals in its place. Not only did this help to identify

the optimal conditions in which crystals will grow, but it helped to find no growth, nucleation,

nucleation transitioning to growth and precipitation.

Methods

In order to optimize the growth of the crystals, two different precipitating agents were

utilized in each of the trials. This was done so that the best precipitating agent was found in order

to get the best crystals. Two trials were performed so that the desired concentrations of the

protein and the salt would be found along with the best precipitating agent that could be pairedwith those ideal concentrations. Trial 1 utilized NaCl and NaI as the precipitating agents. Trial 2

used NaCl and NaBr as the precipitating agents. In order to get the desired crystals, a crystalizing

tray was prepared.

Preparation of Crystal Tray

A 24-well crystal tray was obtained, but only 12 of the wells were utilized. This was so

that the wells could be staggered and

ensured that the coverslips did not

overlap each other. This was so that thegrease applied around the wells created a

tight seal with the coverslips. This

method of using every other well can be

seen in Figure 1.



Lysozyme Crystals 2

In each well, a reservoir was made at the bottom with a combined volume of 0.5mL. The

contents of the reservoirs contained the precipitant solution (NaCl (2M), NaI (2M), NaBr (2M)),

the sodium acetate buffer (1M, pH 4.5), and nanopure water. The concentration each of these

solutions were determined using the ranges in Table 1. After the concentrations were chosen for

each of the components, the concentrations were calculated using Equation 1 (C1V1=C2V2) from

the stock solutions given. The concentrations chosen for trial 1 and trial 2 can be seen in Table 2and Table 3. Then grease was used to rim the top of each of the wells.

After the reservoirs were completed, the drops that hung from the cover slips were made.

Each of the drops contained ½ protein solution (3uL) and ½ reservoir solution (3uL). The

concentration of each of these protein solutions were chosen using the ranges in Table 1 and

calculated using Equation 1. The drops were added to each of the coverslips. Tweezers were

used to grab the corner of the coverslips, quickly flipped over, and placed on top of each well.

This ensured that the drops did not slide off of the coverslips. The tweezers were then pushed

down onto the coverslip to create as seal with the grease on top of each well. The conditions for

trial 1 can be seen in Table 3 (NaCl) and Table 4 (NaI). The conditions for trial 2 can be seen in

Table 5 (NaCl) and Table 6 (NaBr).

Optimization of Crystals

The wells were then left and crystals were allowed to grow over time. The crystals were

checked as soon as possible so that the crystals did not re-dissolve and different crystals grew

instead. After crystals formed, they were put under the microscope. The crystals were stained

with a protein stain in order for the crystals to enhance the images of the crystals under the

microscope. The resulting crystals can be seen in Figure 3 and Figure 4. The results of the

crystal growth can be seen in Table 7. In order to better optimize the crystals, the data for the

crystal growth was collected from the whole class. The results can be seen for NaCl (Graph 1),

NaI (Graph 2), NaNO3 (Graph 3), and NaBr (Graph 4).

Results

Stock solutions used



Lysozyme Crystals 3

Conditions for Trial 1

Conditions for Trial 2



Lysozyme Crystals 4

Resulting lysozyme crystal growth for Trial 1

Resulting lysozyme crystal growth for Trial 2



Lysozyme Crystals 5

Crystal growth results

Class lysozyme growth results



Lysozyme Crystals 6

Discussion

In this lab, we performed 2 trials to determine the best conditions for the ideal crystalsthat we want for x-ray crystallography. The conditions that stayed the same for all of the trials

was the amount of acetate buffer that was used. This was done to make the conditions consistent

for all of the trials.

For trial 1, the precipitant concentration that was used for the wells of NaCl was

configured using the range from Table 1. Each well of NaCl had a different concentration of

precipitant solution from the lowest end of the range to the highest end of the range from 0.4M-

1.2M. This was done so that the right concentration of the NaCl could be chosen. In order to do

this, the protein concentration used was the middle of the range of the protein of concentration

shown in Table 1 from 20mg/mL-60mg/mL. This was done at a 30 mg/mL protein concentration

so that the protein concentration would not be too high or too low. The resulting crystals inFigure 3 were small, so they were not the ideal crystals needed.

The precipitant concentration used for each well of NaI in trial 1 was different for each

well using the range in Table 1. The range used was 0.10M-0.40M with concentrations from the

lowest end to the highest end of the range. The protein concentration used for NaI was 40mg/mL

because this concentration was in the middle of the range of protein concentration in Table 1.



Lysozyme Crystals 7

There were no resulting crystals in trial 1 for NaI, because mold had grown instead. This negated

NaI as an ideal precipitant solution that would be used for x-ray crystallography.

For trial 2, we used NaCl and NaBr. We used NaCl because it produced good crystals

from trial 1, but the crystals were too small. Trial 2 was used as a way to optimize the crystal

growth of NaCl. NaCl in the previous trial had small crystals. For this trial, the concentration ofthe precipitation solution stayed the same for NaCl for trial 2. The change in the NaCl wells were

changed by using the lowest amount of protein concentration in the range for NaCl which is

20mg/mL for half of the NaCl wells. The other half of the wells of NaCl used 60mg/mL. The

reasoning behind this was that the protein concentration could be optimized by seeing if a high

concentration or a low concentration of protein would be ideal. This would allow for larger

crystals. The resulting crystals seen in Figure 4 are much larger than the crystals in trial 1 in well

4 and well 5. This could mean that a larger protein concentration would produce a larger crystal.

The second precipitant in trial 2 was NaBr. This was decided because NaBr produced

mold instead of crystals. The precipitant concentration used for each well of NaBr in trial 2 was

different for each well using the range in Table 1. The range used was 0.30M-0.60M withconcentrations from the lowest end to the highest end of the range. The NaBr wells were

changed by using the lowest amount of protein concentration in the range for NaBr which is

50mg/mL for half of the NaBr wells. The other half of the wells of NaBr used 80mg/mL. This

allowed us to see if the crystals formed from the NaBr wells would be larger with a smaller or

larger protein concentration. The resulting crystals were large as well in NaBr as seen in Figure

4 for well 1, well 2, and well 5. Well 5 had only one crystal, but well 1 and well 2 had larger

crystals. This could mean that a smaller protein concentration for NaBr would produce larger

crystals.

When all of the data was compiled from the whole class, it displayed which of the

precipitants would produce the best crystals as well as which protein concentration wouldenhance that. NaI had no growth for the whole class. NaNO3 produced very little crystals. The

best crystals were seen in NaBr and NaCl. For NaBr, the best conditions seem to be a protein

concentration of ~80mg/mL and a precipitant concentration of ~0.4M. For NaCl, the best

conditions seem to be a protein concentration of ~60mg/mL and a precipitant concentration of

~1M.

Lysozyme Crystals Lab Report Chem 351

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