Crystallography Homework

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University of Houston *

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3201

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Chemistry

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Apr 3, 2024

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docx

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Crystallography Homework The purpose of this assignment is to familiarize you with some of the early crystallography literature. You will use the paper “ Direct Crystallization of Lysozyme from Egg White and Some Crystalline Salts of Lysozyme” by Alderton and Fevold and your lab manual to answer the questions below. Use the questions and the article as a guide for designing your own crystallography experiment (next week’s lab). The crystallography lab you will do in class is more difficult than most you will do in this course. I am not providing you with step-by-step instructions on how to design your experiment. If you look in your manual, you have guidelines and some boundaries, but your group will need to decide what to do based on the journal article and material in the manual. This is what REAL scientists do. You do not make exciting new discoveries following a “cookbook” recipe that has an expected result and outcome. Those types of labs are designed to teach you how to use the equipment and do the math. They do not teach you the critical thinking skills you need to be successful at the research bench. As a research scientist, YOU will write the protocols. YOU make the decisions on HOW and WHY you are going to set up your experiment a certain way. This lab is designed to introduce you to the fundamentals of crystallography and to give you some practice at experimental design. 1. What is lysozyme? Where is it found? What is its’ function? - It is a protein that is found in plant and animal cells. It serves to weaken bacterial cell walls and also prevents bacterial infection of nasal passages and conjunctiva around the eye. 2. According to the abstract at the beginning of the journal article, at what pH range did Alderton and Fevold successfully crystallize lysozyme? What reason do they give as the likely cause of this result? - The pH range was 9.5 to 11.0. Likely caused because of lower solubility of the crystalline product in that region. 3. In the journal article, 5% NaCl solution was used to crystallize lysozyme. The FW for NaCl is 58.44 g/mol. What is the molarity of the NaCl in the solution? - 50 grams / 58.44 = 0.855 M

4. How many mg/ml lysozyme was used by Alderton and Fevold when crystallizing the iodide or bromide salt of lysozyme? Simplify the fraction please - 200mg / 10ml reduces to 20 km/m3 5. Look at Table II on page 5 of the journal article. a. Describe the effect of salt concentration on crystallization. -as salt concentration increased so does the yield of crystallized lysozyme increase b. Describe the effect of pH on crystallization. - as pH increases, the yield of crystallized lysozyme decreases c. Describe the effect of temperature on crystallization. - as temperature increases, the yield of crystallized lysozyme increases then decreases. 6. Look at page 89-94 of your manual. Your group will designing an experiment to test conditions of crystallization of lysozyme. Your team will set up between 6 to 12 wells in a crystallography plate. One of your wells must be a control. Review the materials that will be provided (page 90). You can dilute any of the materials supplied but you cannot go to higher concentrations. Propose an experiment. You may test crystallization in different buffers, vary the NaCl level, vary lysozyme concentration, or vary polyethylene glycol (PEG) concentration. PEG is a crowding agent though to encourage crystallization by forcing the protein molecules closer together in solution. You do not need to work out the protocol details in this assignment. You will do this in class. What I would like to see here is a summary of what your control condition will be and how the conditions in the other 11 wells will vary. Remember, you can only change one variable at a time or you will not know which of the changes you made resulted in crystallization. Write what conditions you would like to test in each circle on the crystallography plate diagram below. Each circle represents a well on the plate.

Well 9: 10% NaCl, 60%, PEG, acetate buffer pH 4.8 Well 8: 25% NaCl, 15% PEG, 0.05 buffer pH 4.8 Well 7: 1.5 M NaCl, 15% PEG in 0.05 buffer pH 4.8 Well 6: 4 M NaCl, 30 % PEG in buffer pH 4.5 Well 5: 3 M NaCl, 0.05 acetate buffer pH 4.8, 0.05 acetate Well 4: 2 M NaCl, 20% PEG in 0.05 M buffer pH 4.8 Well 3: 5 m NaCl, 20% PEG in 0.05 M buffer pH 4.5 Well 2: 1 M NaCl , 50% PEG in 0.05 M sodium acetate pH 4.5 Well 1: 5 M NaCl + 0.05 acetate buffer pH 4.8 Control: 1 M NaCl + 20% PEG in 0.05 acetate pH 4.8 1 2 A B 6 4 3

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