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Intro to structural biology

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Introduction to protein structure and structural biology techniques to study structure/function relationships, with an emphasis on x-ray crystallography.

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Intro to structural biology

  1. 1. Structural biology & why it’s awesome Bri Bibel Aka the bumbling biochemist
  2. 2. • what is structural biology? • why is it important? • how do we do it?
  3. 3. Structural biology • THE WHAT: A scientific discipline that looks at the molecular structure of biological macromolecules and how that STRUCTURE relates to its FUNCTION • THE WHY: Answers questions like: • Why do molecules work the way they do? • What specifically makes one (or a group of them) well-suited for a particular task? • Can we manipulate them to work even better or do other things? • THE HOW: • incorporates principles and techniques of: MOLECULAR BIOLOGY BIOCHEMISTRY BIOPHYSICS
  4. 4. • what is structural biology? • why is it important? • how do we do it?
  5. 5. Structure & function are intimately connected We can exploit this relationship to learn about function from structure and structure from function
  6. 6. What do we mean by structure? Primary structure secondary structure tertiary structure quaternary structure Proteins have multiple layers of structure underlying their final 3D shape
  7. 7. But where does this structure come from… DNA contains the instructions to make amino acids, which are a protein’s building blocks R
  8. 8. AMINO ACIDS Amino acids are the building blocks of proteins & there are 20 common ones They all have the same generic backbone HYDROGEN NITROGEN CARBON OXYGEN
  9. 9. • But they have unique side chains (aka R groups) R AMINO ACIDS
  10. 10. AMINO ACIDS Different side chains have different properties SMALL & FLEXIBLE BIG & BULKY POSITIVELY CHARGED NEGATIVELY CHARGED RWATER-LOVING HYDROPHILIC WATER-AVOIDING HYDROPHOBIC
  11. 11. AMINO ACIDS The side chains’ properties influence how the proteins fold put us next to each other put me at the surface hide me in the middle I don’t bend that way… Don’t expect me to stay still! SMALL & FLEXIBLE BIG & BULKY POSITIVELY CHARGED NEGATIVELY CHARGED WATER-LOVING HYDROPHILIC WATER-AVOIDING HYDROPHOBIC
  12. 12. PRIMARY STRUCTUREAmino acids link together to form polypeptide chains - this is your PRIMARY STRUCTURE A protein’s gene contains the instructions for what order to put them in
  13. 13. SECONDARY STRUCTUREInteractions between the backbone leads to SECONDARY STRUCT can maximize favorable interactions by folding into a couple common motifs alpha helix (α helix) beta strands
  14. 14. TERTIARY STRUCTUREInteractions between the side chains within the same chain lead to TER leads to intricate overall shape of the chain
  15. 15. QUARTERNARY STRUCTURE Some proteins are made up of multiple chains, and interactions between the side chains of different chains lead to QUATERNARY STRUCTURE
  16. 16. What does the structure “do”?
  17. 17. x-ray crystallography nuclear magnetic resonance (NMR) electron microscopy LOOK AT ITS STRUCTURE CHANGE IT TEST ITS FUNCTION binding assays activity assays site-directed mutagenesis; truncations What’s it “supposed” to do? How can we measure that? Can it still do what it’s “supposed to” do? Can it do new things?
  18. 18. Thought exercise • Suppose I tell you what an object does and ask you how it works • a structural biologist will want to know what it looks like. Why? • Consider the converse: I show you an object and ask you what it does
  19. 19. Specific functions are often carried out by specific parts DOMAINS open a beer bottle uncork a bottle of wine slice open an envelope
  20. 20. This is true for proteins too P O H 3’ 5’ O H P 3’ 5’ open a beer bottle uncork a bottle of wine slice open an envelope But they often face more difficult problems… PNKP DNA Ligase
  21. 21. What parts do what? P O H O H P 3’ 5’ 3’ 5’ Polynucleotide kinase phosphatase (PNKP) Bernstein et al., Molecular Cell, 2005
  22. 22. How do we know?
  23. 23. I wonder what that does… Structural biologists use mutations to examine function
  24. 24. Mutations to different parts can have different effects that can tell us about what that part does
  25. 25. We can do something similar with proteins • But we need protein… • We can introduce the gene for the protein into bacteria or insect cells • We grow those cells & those cells make lots of the protein, which we can purify
  26. 26. Since we’re introducing the gene, we have the opportunity to make changes to it… Control the gene, control the protein… Changes to the gene change the primary structure, which can then affect the higher structural levels & perhaps the function
  27. 27. We can add a specific sequence of amino acids to act as a “tag” so we can purify it more easily Control the gene, control the protein…
  28. 28. Control the gene, control the protein… We can mutate specific amino acids to test for function SITE-DIRECTED MUTAGENESIS This can identify “active sites” where the action happens and/or binding sites for other molecules
  29. 29. Control the gene, control the protein… We can truncate, or shorten, the ends, or delete pieces from the middle This can help with crystallization, as we’ll see later…
  30. 30. A more biological example P O H P P 3’ 5’ 3’ 5’ Polynucleotide kinase phosphatase (PNKP)
  31. 31. A more biological example P O H O H O H 3’ 5’ 3’ 5’ Polynucleotide kinase phosphatase (PNKP)
  32. 32. A more biological example P O H O H P 3’ 5’ 3’ 5’ Polynucleotide kinase phosphatase (PNKP)
  33. 33. Choose your readout carefully… P O H O H P 3’ 5’ 3’ 5’ An experiment that only looks at whether the DNA gets fixed wouldn’t be able to tell you which step was blocked
  34. 34. If we know what parts do what, we can use structure-guided design… • What if you want to open your bottles without worrying about cutting your finger?
  35. 35. If we know what parts do what, we can use structure-guided design… • used to develop protease inhibitors for HIV https://www.sciencedirect.com/science/article/pii/S0022283617303157
  36. 36. In addition to what does what, you can figure out “how” it does it Location, location, location
  37. 37. In addition to what does what, you can figure out “how” it does it Not all mutations are created equal
  38. 38. Changing existing molecules • Instead of adding a shield, just remove the blade
  39. 39. Changing existing molecules • you can buy mutants of T4 PNK that have kinase activity, but no phosphatase activity • great for radiolabeling RNA so you can track it!
  40. 40. In addition to what does what, you can figure out “how” it does it Integrate information from different types of experiments
  41. 41. Designing new molecules What if you also want to be able to file your nails? MIX & MATCH!
  42. 42. Structure-guided recombination http://fhalab.caltech.edu/?page_id=171 Frances Arnold 2018 Nobel Laurette in Chemistry! use knowledge of structure to figure out how to put pieces of different proteins together to mak Linus Pauling Professor of Chemical Engineering, Bioengineering and Biochemistry CalTech
  43. 43. Not so fast…you need a structure!
  44. 44. • what is structural biology? • why is it important? • how do we do it? • what is structural biology? • why is it important? • how do we do it?
  45. 45. Getting to a structure • a few key methods: • x-ray crystallography • cryo-electron microscopy (cryo-EM) • nuclear magnetic resonance (NMR)
  46. 46. Getting to a structure Jones, Nature, 2014 https://www.nature.com/news/crystallography-atomic-secrets-1.14608 Crystallography
  47. 47. Getting crystals • hanging-drop diffusion drop of protein + “magic liquid” “magic liquid” only
  48. 48. But getting crystals is rarely easy… • to crystallize, proteins must “freeze” in a precisely ordered manner • but what if there’s a “loose screw”? Proteins move around and can exist in different conformations Not all of which are equally informative…
  49. 49. Finding the right conditions: screening
  50. 50. Flexible regions can be problematic… • flexible regions can prevent crystallization • if only 1 part’s causing problems, you can try removing it
  51. 51. Look at the pieces separately
  52. 52. Look at the pieces separately Bernstein et al., Molecular Cell, 2005
  53. 53. Get help from a homolog! • sometimes similar proteins from the same species or other species crystallize more easily this is actually the murine (mouse) version can be closely related can be more distantly related they can have very different sequences but similar structures
  54. 54. Try another method cryo-electron microscopy (cryo- EM) https://cryoem.slac.stanford.edu/what-is-cryo-em instead of trying to capture them in a single conformation, let them move around, then take a snapshot and pick out the most prominent ones group together & average the ones that look similar good for BIG things
  55. 55. nuclear magnetic resonance (NMR) Let it move & look at it all while it moves! Good for small, flexible, things use a strong magnet to alter the magnetic field and see how the nuclei of the atoms in the proteins respond gives you an “ensemble” of images https://slideplayer.com/slide/6420286/

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