1. 1 Levels of Protein Structure Primary to Quaternary Structure

2. 2 Learning Objectives  After today you should be able to: –Define the structural levels of proteins. –Identify the structural units of the protein backbone. –Explain why some backbone conformations are “forbidden”, i.e. not found in natural proteins. –Name properties on which the amino acids can be grouped. –Name more amino acids than you could before One and three letter codes

3. 3 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

4. 4 L-amino acids in Living organisms N: 14 C: 12 O: 16

5. 5 How to Group Them?  Many features –Charge +/- Acidic vs. basic (pK a ) –Polarity (polar/non-polar) Type, distribution –Size Length, weight, volume, surface area –Type (Aromatic/aliphatic)

6. 6 The Amino Acids Asn (N) Asp (D) Gln (Q) Glu (E) His (H) Lys (K) Arg (R) Tyr (Y) Trp (W) Phe (F) Gly (G) Pro (P) Ile (I) Met (M) Leu (L) Ala (A) Val (V) Ser (S) Cys (C) Thr (T)

7. 7 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

8. 8 Blosum62 substitution matrix

9. 9 The Evolution Way  Based on Blosum62 matrix  Measure of evolutionary substitution probability

10. 10 The Simple Aliphatic Ile (I) Met (M) Leu (L) Val (V) Ala (A)

11. 11 The Small Polar Thr (T) Cys (C) Ser (S) Cystin

12. 12 The Unusual  P, G  Also aliphatic  Structural impact  Strictly speaking, proline is an imino acid Gly (G) Pro (P)

13. 13 The Acidic and Their Derivatives Asn (N)Asp (D) Gln (Q)Glu (E)

14. 14 The Basic His (H)Lys (K)Arg (R)

15. 15 The Aromatic Tyr (Y) Trp (W) Phe (F) His (H)

16. 16 Proteins Are Polypeptides  The peptide bond  A polypeptide chain

17. 17 Torsion Angles

18. 18 Ramachandran Plot  Allowed backbone torsion angles in proteins N H

19. 19 Small Exercise (5 minutes)  For the polypeptide on the left discuss the following with your neighbour: –Why is the lower right quadrant a ”forbidden” region in the Ramachandran plot? –What makes Gly a special amino acid when it comes to Ramachandran plots? –What about Pro?

20. 20 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…

21. 21  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.     

22. 22 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

23. 23 Hydrophobic vs. Hydrophilic  Globular protein (in solution)  Membrane protein (in membrane) MyoglobinAquaporin

24. 24 Hydrophobic vs. Hydrophilic  Globular protein (in solution)  Membrane protein (in membrane) MyoglobinAquaporin Cross-section

25. 25 Characteristics of Helices  Aligned peptide units  Dipolar moment  Ion/ligand binding  Secondary and quaternary structure packing  Capping residues  The  helix (i→i+4)  Other helix types! (3 10,  ) N C

26. 26 Helix Types

27. 27 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

28. 28  -Sheets  Multiple strands  sheet –Parallel vs. antiparallel –Twist –Coil regions between the secondary structure elements Thioredoxin

29. 29  -Sheets  Multiple strands  sheet –Parallel vs. antiparallel –Twist

30. 30  -Sheets  Multiple strands  sheet –Parallel vs. antiparallel –Twist

31. 31  -Sheets  Multiple strands  sheet –Parallel vs. antiparallel –Twist

32. 32  -Sheets  Multiple strands  sheet –Parallel vs. antiparallel –Twist  Strand interactions are non-local  Flexibility –Vs. helices –Folding AntiparallelParallel

33. 33 Summary  Amino acids in Living organisms have L- configuration  One & three letter codes  Groups of amino acids: hydrophobic...  The backbone of polypeptides form regular secondary structures. –Helices, sheets & loops.

34. 34 Building Blocks NH 2 /NH 3 + S/SH Guanidine Imidazole Phenyl Indole CC H-bond Amide C/CH/CH 2 /CH 3 COOHOHPeptide unit (amide) N C O N C O

35. 35 Building Blocks N O C CC CC CC CC SS Methionine Single amino acid Residue N O C CC O N O C CC O n-1 N n+1 C n-1      O

36. 36 Procedure 1.Build backbone in extended conformation (strand). 2.Twist to align all peptide units (look at the C=O groups). 3.Add H-bonds (i+4) to construct an ideal  - helix. 4.Add side chains along the way (pointing “down”). N C