1. PROTEIN STRUCTURE AND FUNCTION

2. Proteins Are Where It’s At Proteomics Gene regulation Drug Discovery Understanding evolution Etc.

3. Proteins are Where It’s Been Enzymes  ß-galactosidase Antibodies  Anti-Hepatitis B Hormones  Human Growth Hormone  Estrogen  Testosterone

4. Proteins are Where It’s Been Structural proteins  Collagen Transportation  Haemoglobin

5. Proteins Are Us In cells, when something needs to be done, it is a protein that does it. The human body contains over 30,000 different types of protein Other organisms have many of the same proteins as well as different ones Enzymes are the biggest class  3,000 enzymes in average mammalian cell  ß-galactosidase is an enzyme

6.  Transcription Factors  Control expression of genes  Hormones  Control body function  Antibodies  Fight infection  Enzymes  Speed up chemical reactions  Carrier molecules  Haemoglobin - carries oxygen in the blood Classes of Proteins Structural  Collagen  Found in bone and skin  Keratin ■ Makes hair and nails  Fibrin  Helps clot blood  Elastin  Major part of ligaments

7. Proteins Are Diverse In Structure Proteins can do many things because they are structurally diverse Differ in many properties:  Size  Shape  Charge distribution  Hydrophobicity  Solubility properties

8. Variability Comes From Amino Acids Are polymers composed of 20 different amino acid building blocks As letters can be arranged in many ways, so too can amino acids Number, type and arrangement of amino acids determines structure and function Insulin has about 50 AA  Most are >> bigger - from 100s to 1000s  Allows for great diversity

9. Amino Acids All amino acids have a carboxyl group and an amino group A different R group is attached to each amino acid

10. Amino Acid

11. R groups make each amino acid different Some are:  Polar  Nonpolar  Charged  Acids  Bases

12. Twenty Amino acids

13. Folding DNA always has same structure But proteins fold into many different shapes Folding depends ultimately on amino acid composition Structure of proteins determines function Structure allows proteins to  BIND to other molecules  RECOGNIZE other molecules

14. Protein Structure And Function

15. Shape Is Critical Change in one amino acid can change the structure of the protein with a large effect on function  Sickle cell anemia

16. Image from Medline Plus Sickle Cell Anemia Single DNA base pair is mutated Therefore one amino acid is altered Glutamic acid is switched to valine Glutamic acid is negatively charged, valine is neutral Changes how hemoglobin packs in cells Alters shape of red blood cells when oxygen is low.

17. Levels of Protein Structure Protein structure is complex and important, so it is classified into:  1° - Primary  2°- Secondary  3° -Tertiary  4°- Quaternary

18. Primary Structure Linear sequence of amino acids Peptide bond (covalent bond) holds it together Beads on a string

19. Primary Structure

20. Peptide Bonds R O H R l ll l l NH 2 –C – C – 0 –H + H –N – C—COOH l l H H  R O H R l ll l l NH 2 -- C –C – N – C – COOH + H 2 O l l H H

21. Secondary Structure Regular repeating patterns of twists or kinks of the amino acid chain Examples  Alpha helix  Beta pleated sheet

22. Secondary Structure Hydrogen bonds hold structure together  Weak, noncovalent, molecular interactions Hydrogen atom is bonded to an electronegative atom (like F, O, N) that is also partially bonded to another atom (usually also F, O, N)

23. Figure from National Human Genome Research Institute, by artist Darryl Leja. Used with permission.

24. Tertiary Structure 3° 3-D Globular Configuration formed by bending and twisting of the polypeptide chain Stabilized by:  Hydrogen bonds  Electrostatic interactions 1.(Positive and negative)  Hydrophobic interactions  Sometimes covalent bonds 1.Disulfide bonds

25. Quaternary Structure 4° Two or more polypeptide chains associate with each other

26. ß-Galactosidase Link to Protein Data Bank for picture of molecular image of ß-galactosidase www.pdb.org

27. Higher Order Structure Higher order (secondary, tertiary, quaternary) structure is relatively “weak” In nature, “weakness” of noncovalent interactions is important  Flexibility 1.Enzymes change shape when binding to their substrates 2.Necessary for proper function

28. How Proteins Lose Normal Structure And Function Primary structure hard to disrupt; covalent bonds are strong

29. How Proteins Lose Normal Structure And Function Can be broken apart by enzymes (proteases) that digest the covalent peptide bonds  Called proteolysis  Occurs naturally in digestion  Can be a problem in the lab; proteases can destroy protein of interest  Use cold to avoid proteolysis

30. How Proteins Lose Their Structure And Function Sulfur groups on cysteines may undergo oxidation to form disulfide bonds that are not normally present Proteins can aggregate leading to precipitation Proteins can adsorb (stick to) surfaces

31. Higher Order Structure In Lab Loss of higher order structure is denaturation Denaturation occurs fairly easily  Affected by changes in pH  Ionic strength  Temperature May or may not be reversible

32. Denaturation

33. Manipulating Higher Order Structure Often manipulated in lab  Destroy folding when we do PAGE  Use buffers to maintain the structure  Use cold temperatures  Add reducing agents to prevent unwanted disulfide bonds in the lab --DTT or  -ME

34. Analyzing Protein Structure X-Ray Crystallography  Like a CAT scan in medicine  X-ray taken at multiple angles and computer uses the data to calculate a 3D image Nuclear Magnetic Resonance  Like an MRI in medicine

35. X-ray Crystallography Isolate and purify protein Form a crystal of the protein  Molecules of the protein are arranged in an orderly lattice 1.Dissolve protein in solvent 2.Precipitate into a crystal X-Ray the crystal

36. X-ray Crystallography Analyze diffraction pattern using software  Make electron density map Process used to take years  Different versions of crystal for comparison 1.Each with different heavy metal in lattice to provide reference point New X-ray sources - synchrotrons have reduced data collection time to few days

37. X-ray Crystallography Synchotron - Argonne National Lab Still takes weeks to go from gene sequence to 3-D structure (mrsec website, nanotechnology at UW- Madison)

38. Structural Genomics Goal to solve thousands of structures a year Large scale automation required Syrrx - structural genomics company  Robot places a drop of protein into 480 wells  11,000 crystallization experiments in 24 hours  New robot - 130,000 a day

39. Nuclear Magnetic Resonance Similar to MRI - Magnetic Resonance imaging in medicine No need for crystals, proteins in solution Works for relatively simple proteins