1. Protein Structure Elements Primary to Quaternary Structure

2. Learning Objectives After this lesson you should be able to: –Define the structural levels of proteins. –Identify regular secondary structure elements. –Identify the structural units of the protein backbone. –Explain why some backbone conformations are favoured and some are “forbidden” (not found in natural proteins). –Name properties on which the amino acids can be grouped. –Explain the driving forces behind protein folding related to the properties of the backbone and the side chains.

3. Proteins Are Polypeptides The peptide bondA polypeptide chain

4. Structure Levels Primary structure = Sequence (of amino acids) Secondary Structure = Helix, sheets/strands, bends, loops & turns (all defined by H-bond pattern in backbone) Structural Motif = Small, recurrent arrangement of secondary structure, e.g. –Helix-loop-helix –Beta hairpins –EF hand (calcium binding motif) –Many others… Tertiary structure = Arrangement of Secondary structure elements within one protein chain MSSVLLGHIKKLEMGHS…

5. Myoglobin Haemoglobin Quaternary Structure Assembly of monomers/subunits into protein complex –Backbone-backbone, backbone-side-chain & side-chain-side-chain interactions: Intramolecular vs. intermolecular contacts. For ligand binding side chains may or may not contribute. For the latter, mutations have little effect.     

6. A Bit About Protein Folding How and why proteins fold

7. Why Fold? Hydrophobic collapse –Hydrophobic residues cluster to “escape” interactions with water. –Polar backbone groups form secondary structure to satisfy hydrogen bonding donors and acceptors. –Initially formed structure is in molten globule state (ensemble). –Molten globule condenses to native fold via transition state

8. Hydrophobic Core Hydrophobic side chains go into the core of the molecule – but the main chain is highly polar. The polar groups (C=O and NH) are neutralized through formation of H-bonds. Myoglobin SurfaceInterior

9. Hydrophobic vs. Hydrophilic Globular protein (in solution) Membrane protein MyoglobinAquaporin

10. Hydrophobic vs. Hydrophilic Globular protein (in solution) Membrane protein MyoglobinAquaporin Cross-section

11. From Unfolded to Native State  G =  H - T×  S -------------------------  G: Free (Gibbs) energy  H: Enthalpy (interactions)  S: Entropy (conformations/ states) E U F T GG Unfolded state, ensemble Native fold, one structure Transition state, one or more narrow ensembles

12. Protein Stability & Dynamics Folded proteins are: –Only marginally stable (enthalpy and entropy almost balance at physiological temperatures) Allows for easy degradation and reuse. Amyloid exception. –Dynamic “Breathing” motions on pico- to nanosecond scale. Allows substrates/products to enter/leave enzymes. Allows allosteric regulation of activity.

13. Amino Acids Proteins are built from amino acids Amino group and acid group Side chain at C  Chiral, only one enantiomer found in proteins (L-amino acids) 20 natural amino acids N O C CC CC CC CC SS Methionine

14. Amino Acid Properties Many features –Charge +/- Acidic vs. basic (pK a ) –Polarity (polar/non-polar) Type, distribution –Size Length, weight, volume, surface area –Type (Aromatic/aliphatic)

15. Grouping Amino Acids Livingstone & Barton, CABIOS, 9, 745-756, 1993 A – Ala C – Cys D – Asp E – Glu F – Phe G – Gly H – His I – Ile K – Lys L – Leu M – Met N – Asn P – Pro Q – Gln R – Arg S – Ser T – Thr V – Val W – Trp Y - Tyr

16. The Evolution Way Based on Blosum62 matrix Measure of evolutionary substitution probability

17. Backbone Properties Amide bond planarity2 degrees of rotational freedom per residue

18. Ramachandran Plot Allowed backbone torsion angles in proteins N H Residue Peptide bond

19. Torsion Angles

20. Characteristics of Helices Backbone interactions are local Aligned peptide units  Dipolar moment N C

21. Helix Types

22.  -Sheets Multiple strands  sheet –Parallel vs. antiparallel –Twist Strand interactions are non-local Flexibility –Vs. helices –Folding AntiparallelParallel

23.  -Sheets Thioredoxin

24.  -Sheets Thioredoxin

25.  -Sheets Thioredoxin

26.  -Sheets Thioredoxin

27. Not All  -Sheets Are Flat NitrophorinThioredoxin

28. Residue Patterns Helices –Helix capping –Amphiphilic residue patterns Sheets –Amphiphilic residue patterns –Residue preferences at edges vs. middle Special residues –Proline Helix breaker –Glycine In turns/loops/bends N C

29. Turns, Loops & Bends Revisited Between helices and sheets On protein surface Intrinsically “unstructured” proteins

30. Summary The backbone of polypeptides form regular secondary structures. –Helices, sheets, turns, bends & loops. These are the result of local as well as non- local interactions. Secondary structure elements are associated with specific residue patterns.

31.  -sheet and  -helices  -sheet  -helix 1M8N TheoreticalReal