Young Scholars Program Sponsored by: Dr. Thayumanasamy Somasundaram

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1 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

2 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 2

3 chicken (Gallus gallus domesticus) eggs, 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, three biophysical tests were performed (circular dichroism, UV- visual spectrophotometer, and fluorospectrophotometer) prior to actually crystallizing the protein to confirm the source of was actually uncontaminated. 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. 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 in buffer at various concentrations (45, 40, 30, 20, 15, 10)mg/mL, made one 3

4 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 solution + 2 μl buffer suspended over 600 μl well solution (10% of the indicated compound dissolved in buffer). 45mg/mL 20mg/mL 10mg/mL 45mg/mL 20mg/mL 10mg/mL 45mg/mL 20mg/mL 10mg/mL 45mg/mL 20mg/mL 10mg/mL 45mg/mL 20mg/mL 10mg/mL 45mg/mL 20mg/mL 10mg/mL 4

5 +NaNO3 + NaNO3 + NaNO3 + NaNO3 + NaNO3 + NaNO3 + NaNO3 + NaNO3 + NaNO3 +KSCN + KSCN + KSCN + KSCN + KSCN + KSCN + KSCN + KSCN + KSCN While the crystals were forming, the biophysical tests were carried out on a single, low concentration of 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: Needle crystal: too fragile to harvest for diffraction, which renders it useless. Good crystal: all in one piece 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. 5

6 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 An unfrozen crystal mounted in a loop crystal and solution to prevent the ice interference 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. 6

7 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: 7

8 Through computer analysis this diffraction confirmed the P symmetry of this crystal that was previously hypothesized, the other diffractions provided conclusive evidence that P1 and P 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 Web. 14 July < PDB. "RCSB PDB." Protein Data Bank. RCSB. Web. 14 July < "X-RAY Crystallography." St. Olaf College A Private Liberal Arts College of the Lutheran Church in Minnesota. St. Olaf College. Web. 14 July < 8

9 Shane Matthews X Ray Crystallography: The Effect of Different Substrates on the Crystalline Structure of Lysozyme by Shane Matthews and Dean Sage Written by Shane Matthews FSU Young Scholars Program, Summer 2011 Sponsor: Dr. Thayumanasamy Somasundaram 1

10 Abstract Shane Matthews 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. 2

11 Shane Matthews 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. 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 3

12 Shane Matthews 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 research and development. 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 concentrations of. 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. 4

13 Shane Matthews In the loop method, a crystal is plucked from the glass slide, flash 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 which were diffracted successfully. 5

14 Shane Matthews 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, and sodium nitrate. 6

15 Shane Matthews 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 7

16 Shane Matthews was produced by the crystal crystallized using NaCl; the resolution of the diffraction is the highest. Cystalline arrangements achieved using different substrates Substrate used during crystallization Space Group NaCl P NaI P1 NaNO3 P1 KSCN P Using four different substrates, three unique space groups of were achieved; this is a perfect example of pseudopolymorphism. Conclusion The protein 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 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 8

17 Shane Matthews structure is very valuable in that it can be used to manipulate molecules for use in pharmaceuticals. 9