Protein Crystal Growth Using Osmotic Dewatering in Microgravity

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1 Protein Crystal Growth Using Osmotic Dewatering in Microgravity Mike Smith ASEN 5519 Space Experiment Technology and Design Image From Final Project ASEN5519 Mike Smith Page - 1

2 Agenda Why grow crystals? How crystals form? Why grow crystals in space? Basics of osmotic dewatering Basic fundamentals Requirements for crystal growth apparatus Existing apparatus Options Control each chamber with valves Make each chamber a different volume Make each chamber a different temperature Comparison of options Design Description Final Project ASEN5519 Mike Smith Page - 2

3 Why grow crystals? Crystals of proteins and nucleic acids are grown to be able to determine their 3-D structure The structure provides valuable insight into the specific biological roles that these molecules play in our body Image from Image from Once a single crystal is grown, its structure can be determined using X-ray diffraction Researchers working to create drugs can use this information to tailor compounds that selectively bind to target sites on the protein and inhibit the activity of the protein Final Project ASEN5519 Mike Smith Page - 3

4 How crystals form? Image From Chemistry and Chemical Reactivity, 2nd Edition Crystals grow as molecules move through a fluid, bump into one another, and latch into position to build a repeating pattern, like a child's building blocks Once a minimum number of molecules have joined together the crystal becomes stable and crystal growth begins The process involves diffusion as molecules migrate from an area of high concentration to an area of low concentration (the depleted area just next to the crystal) Final Project ASEN5519 Mike Smith Page - 4

5 Why grow crystals in space? Large, single crystals are required for X-ray diffraction (0.1 mm in its smallest dimension, preferably 0.2 to 0.4 mm) Protein crystals are very fragile Protein crystals can take weeks to grow on earth if they can be grown at all In a microgravity environment, crystals can be grown that are of higher quality and often much larger than can be grown on earth Both of these crystals were grown and were better than any grown on earth MALIC ENZYME INSULIN Images From Final Project ASEN5519 Mike Smith Page - 5

6 Basics of osmotic dewatering Protein and Salt In Solution with Water Dialysis Bag Chamber Salt (NaCl) in Solution with Water The protein is held in solution in a dialysis bag The chamber is filled with 30% saturated solution of salt The salt concentration in the chamber is slowly raised by pumping in a solution with a higher salt concentration When the salt reaches a high enough concentration (around 55%), the protein molecules are forced out of solution and begin to crystallize Final Project ASEN5519 Mike Smith Page - 6

7 Basic Fundamentals A few definitions: Solution: a homogeneous mixture of two or more substances in a single phase Solute: the material dissolved in a solution Solvent: the medium in which a solute is dissolved to form a solution Saturated Solution: a stable solution in which the maximum amount of solute has been dissolved 35.7 grams of NaCl are required to have a fully saturated solution in 100 ml of H 2 O at 0º C Change in salt concentration can be determined by the following End% = Start% + Injected% - Ejected% Each value has to be calculated by volume Assumption: When calculating the salt concentration in the chamber, it is assumed the salt in the chamber diffuses equally throughout the chamber before more salt solution is pumped in Final Project ASEN5519 Mike Smith Page - 7

8 Basic Fundamentals Amount of salt required for saturation varies with temperature Image From Chemistry and Chemical Reactivity, 2nd Edition Final Project ASEN5519 Mike Smith Page - 8

9 Requirements for crystal growth apparatus Crystal growth apparatus is to provide crystal growth chambers with varying salt concentrations Apparatus to require minimum involvement of astronauts Apparatus to require minimum number of measurements Apparatus to require minimum amount of control Each chamber to begin and end crystal growth at the same time Apparatus to be leak free Final Project ASEN5519 Mike Smith Page - 9

10 Existing apparatus Chamber Apparatus X Y Z M N O P In Pump Out Existing apparatus uses seven chambers connected in series to control salt concentration Each chamber begins with mixture with 30% salt concentration Mixture with 70% salt concentration pumped in Over time, each chamber reaches 70% concentration Each chamber is approximately 1 ml 1 μl of 70% salt concentration is pumped in every 10 seconds Final Project ASEN5519 Mike Smith Page - 10

11 Salt Concentrations using Existing Apparatus Concentration (%) Chamber X Z N Q Y M P Time (hour) Final Project ASEN5519 Mike Smith Page - 11

12 Options Option 1: Control each chamber with valves Pump in 70% salt solution until desired concentration is reached and then close valve Stagger starting times to achieve crystal growth at same time Option 2: Make each chamber a different volume Pump in 70% solution for a set amount of time Chamber with smallest volume will have quickest increase of salt concentration Chamber with largest volume will have slowest increase of salt concentration Stop pumping in 70% solution when desired gradient reached Option 3: Make each chamber a different temperature Amount of salt in saturated mixture varies with temperature Each chamber to have a different temperature Because each chamber has a different temperature, each will have a different concentration Final Project ASEN5519 Mike Smith Page - 12

13 Option 1: Control with Valves - Schematic Apparatus Chamber Outlet X Y Z M N O P Valve Valve Valve Valve Valve Valve Valve Pump Final Project ASEN5519 Mike Smith Page - 13

14 Option 2: Different Volumes - Schematic Apparatus Chamber Outlet X Y O P N M Z Pump Final Project ASEN5519 Mike Smith Page - 14

15 Comparison of Options Option 1: Control with Valves Pros Cons Very precise control of salt concentration Complex (many valves, wiring, controlled by computer) High Cost Requires control of seven different valves Option 2: Different Volumes Pros Cons Fairly precise control of salt concentration Simple No individual control required on each chamber Requires more space to achieve different volumes (1mL vs up to 4 ml) Option 3: Control with Temperature Not feasible with NaCl due to small change in % concentration with change in temperature Final Project ASEN5519 Mike Smith Page - 15

16 Design Description Recommend option 2: different volumes Design features Can obtain a different salt concentration in each chamber at the same time Can vary how long it takes to reach desired salt concentration by varying injection volume or time If desired concentration is reached in 8 hours, design can be sized as follows Sizing assumes 1 μl injected into each cavity every 10 seconds Cavity X Y Z M N P Q Salt Concentration (%) Cavity Volume (ml) Final Project ASEN5519 Mike Smith Page - 16

17 Salt Concentration of proposed apparatus 80 Stop pumping here to freeze concentration 70 Concetration (%) Chamber X Y Z M N P Q Time (hr) Final Project ASEN5519 Mike Smith Page - 17

18 References Internet References Chemistry and Chemical Reactivity, 2nd Edition, John C. Kotz and Keith F. Purcell, Saunders College Publishing, Harcourt Brace Jovanovich College Publishers Final Project ASEN5519 Mike Smith Page - 18