More Related Content
What's hot
What's hot (20)
Similar to Intro to structural biology
Similar to Intro to structural biology (20)
Recently uploaded
Recently uploaded (20)
Intro to structural biology
- 1. Structural biology & why it’s awesome Bri Bibel Aka the bumbling biochemist
- 2. • what is structural biology? • why is it important? • how do we do it?
- 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. • what is structural biology? • why is it important? • how do we do it?
- 5. Structure & function are intimately connected We can exploit this relationship to learn about function from structure and structure from function
- 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. But where does this structure come from… DNA contains the instructions to make amino acids, which are a protein’s building blocks R
- 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. • But they have unique side chains (aka R groups) R AMINO ACIDS
- 10. AMINO ACIDS Different side chains have different properties SMALL & FLEXIBLE BIG & BULKY POSITIVELY CHARGED NEGATIVELY CHARGED RWATER-LOVING HYDROPHILIC WATER-AVOIDING HYDROPHOBIC
- 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. 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. 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. TERTIARY STRUCTUREInteractions between the side chains within the same chain lead to TER leads to intricate overall shape of the chain
- 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. What does the structure “do”?
- 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. 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. Specific functions are often carried out by specific parts DOMAINS open a beer bottle uncork a bottle of wine slice open an envelope
- 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. 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. How do we know?
- 23. I wonder what that does… Structural biologists use mutations to examine function
- 24. Mutations to different parts can have different effects that can tell us about what that part does
- 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. 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. 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. 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. 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. A more biological example P O H P P 3’ 5’ 3’ 5’ Polynucleotide kinase phosphatase (PNKP)
- 31. A more biological example P O H O H O H 3’ 5’ 3’ 5’ Polynucleotide kinase phosphatase (PNKP)
- 32. A more biological example P O H O H P 3’ 5’ 3’ 5’ Polynucleotide kinase phosphatase (PNKP)
- 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. 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. 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. In addition to what does what, you can figure out “how” it does it Location, location, location
- 37. In addition to what does what, you can figure out “how” it does it Not all mutations are created equal
- 38. Changing existing molecules • Instead of adding a shield, just remove the blade
- 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. In addition to what does what, you can figure out “how” it does it Integrate information from different types of experiments
- 41. Designing new molecules What if you also want to be able to file your nails? MIX & MATCH!
- 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. Not so fast…you need a structure!
- 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. Getting to a structure • a few key methods: • x-ray crystallography • cryo-electron microscopy (cryo-EM) • nuclear magnetic resonance (NMR)
- 46. Getting to a structure Jones, Nature, 2014 https://www.nature.com/news/crystallography-atomic-secrets-1.14608 Crystallography
- 47. Getting crystals • hanging-drop diffusion drop of protein + “magic liquid” “magic liquid” only
- 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. Finding the right conditions: screening
- 50. Flexible regions can be problematic… • flexible regions can prevent crystallization • if only 1 part’s causing problems, you can try removing it
- 51. Look at the pieces separately
- 52. Look at the pieces separately Bernstein et al., Molecular Cell, 2005
- 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. 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. 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/
Editor's Notes
- Structural biology can be one of those arcane fields that's difficult to describe and I found myself having difficulty explaining what I was doing and why to family and friends. So I set out on a mission to share the wonders of structural biology and biochemistry in a fun way on social media and a website through a not-always-super "scicomm superhero" alter ego the bumbling biochemist (lab coat cape and all), with the underlying personal mission of teaching myself how to communicate science more effectively. In this talk, I introduce some of the fundamentals of structural biology.
- Structural biology
- At its core, structural biology is about structure, function, and the relationship between the 2 The “macromolecules” we look at are usually looking at proteins or protein/nucleic acid (RNA &/or DNA) complexes.
- When we talk about “structure,” we’re usually referring to the overall 3D structure, but proteins have layers of structure underneath the final product we “see”. This starts with the primary structure, which is the sequence of amino acid “building blocks” whose chemical characteristics influence how the protein folds.
- Structural biologists often start off knowing (at least some of) what a molecule does (thanks to cell biologists, etc.) but we often have to figure out how best to measure those functions in a test tube (in vitro)
- Radiation can cause breaks in DNA. These ends can be stitched back together by DNA ligase, but only if they have the right “caps” - a molecule called polynucleotide kinase phosphatase (PNK) can convert “wrong” caps but it needs to be able to do 3 things - find the site, phosphorylate 5’ ends and dephosphorylate 3’ ends
- PNKP splits these 3 functions among it’s 3 domains. The kinase domain adds phosphate, the phosphatase domain removes that phosphate, and the FHA domain binds to proteins that are part of the DNA repair pathway that have already “scouted out” the break site
- We can use site-directed mutagenesis to change specific parts of the primary structure, which can then affect the higher structural levels & perhaps the function
- We can use site-directed mutagenesis to change specific parts of the primary structure, which can then affect the higher structural levels & perhaps the function
- We can use site-directed mutagenesis to change specific parts of the primary structure, which can then affect the higher structural levels & perhaps the function
- We can use site-directed mutagenesis to change specific parts of the primary structure, which can then affect the higher structural levels & perhaps the function
- If you know the shape of the blade, you can design a shield to cover it
- Structure-guided design is also often used to “fine-tune” hit compounds - see where they bind and how they might be made to bind better (i.e. are there other potential interactions if you added something?)
- You can use site-directed mutagenesis to find out what specific part within a domain is important for different things. If you don’t “hit” a part that’s important for a particular function, that function won’t be affected, even if it’s in the responsible domain
- Different mutations have different effects. Instead of losing function or being “neutral” mutations can modify a molecule’s functional preferences
- Get information however you can, then combine that information to come up with a mechanistic theory
- Frances Arnold won the 2018 Nobel Prize in Chemistry for research on directed evolution, but her lab also studies how to combine pieces of different proteins together to make “chimeras” with cool new functions. She uses knowledge of the structures of the “parent” molecules to know where are good places to cut & paste so that the parts remain functional
- I’m going to focus mainly on crystallography, because it’s what I use
- In x-ray crystallography, you freeze your molecules in an ordered lattice and shoot x-ray beams at it. The beams will scatter when they hit the molecules, producing a pattern of spots that you can then work backwards from to deduce the underlying structure
- You want the protein to come out of solution but in a very orderly fashion… A technique I commonly use is hanging-drop diffusion. Since the concentration of “magic” in the liquid is higher in the reservoir than in the drop with protein, water will evaporate out of the drop, promoting crystal formation
- Flexibility’s great for a lot of things, like linking together domains that need to move independently, but squirming around and crystallizing don’t mix well…
- Of course, you’re not getting the full picture, but it’s better than no picture at all! And it’s only 1 piece of the puzzle - you combine whatever limited data you get with data from activity tests, etc.
- They were able to get a better look at the floppy FHA domain by crystallizing it apart from the other domains (but with a piece of one of its binding partners so we also get information about how it recognizes the target site)