1. Principles of Protein Structure
ACDEFGHIKLMNPQRSTVWY
primary structure
Stop to ask questions: leave a moment for silence.
Take Home Message: define secondary structure
Next part of the talk will deal with what computational tools we can use for secondary structure prediction in proteins

2. Different Levels of Protein Structure
NH2
Lysine
Histidine
Valine
Arginine
Alanine
COOH

3. Common Secondary Structure Elements
The Alpha Helix
Take Home Message: Alpha Helix commonly observed secondary structure in proteins –looks like a corkscrew

4. Properties of alpha helix
3.6 residues per turn, 13 atoms between H-bond donor and acceptor
approx. -60º;  approx. -40º
H- bond between C=O of ith residue & -NH of (i+4)th residue
First -NH and last C=O groups at the ends of helices do not participate in H-bond
Ends of helices are polar, and almost always at surfaces of proteins
Always right- handed
Macro- dipole

5. Alpha Helix

6. Helical wheel
Residues i, i+4, i+7 occur on one face of helices, and hence show definite pattern of hydrophobicity/ hydrophilicity

7. Introduction to Molecular Biophysics
Association of helices: coiled coils
Introduction to Molecular Biophysics
These coiled coils have a heptad repeat abcdefg with nonpolar residues at
position a and d and an electrostatic interaction between residues e and g.
Isolated alpha helices are
unstable in solution but are
very stable in coiled coil
structures because of the
interactions between them
The chains in a coiled-coil have
the polypeptide chains aligned
parallel and in exact axial
register. This maximizes
coil formation between chains.
The coiled coil is a protein motif that is often used to control oligomerization.
They involve a number of alpha-helices wound around each other in a highly
organised manner, similar to the strands of a rope.

8. Introduction to Molecular Biophysics
The Leucine Zipper Coiled Coil
Introduction to Molecular Biophysics
Initially identified as a structural motif in proteins involved in eukaryotic
transcription. (Landschultz et al., Science 240: (1988).
Originally identified in the liver transcription factor C/EBP which has a Leu
at every seventh position in a 28 residue segment.

9. Association of helices: coiled coils
The helices do not have to run in the same direction for this type of
interaction to occur, although parallel conformation is more common.
Antiparallel conformation is very rare in trimers and unknown in
pentamers, but more common in intramolecular dimers, where the two
helices are often connected by a short loop.
Chan et al., Cell 89, Pages

10. Basis for the helical dipole
In an alpha helix all of the peptide
dipoles are oriented along the
same direction.
Consequently, the alpha helix has
a net dipole moment.
Since the dipole moment of a peptide bond is 3.5 Debye units, the alpha
helix has a net macrodipole of:
n X 3.5 Debye units (where n= number of residues)
This is equivalent to 0.5 – 0.7 unit charge at the end of the helix.
The amino terminus of an alpha helix is positive and the
carboxy terminus is negative.

12. Structure of human TIM
Two helix dipoles are seen to play important roles:
Stabilization of inhibitor 2-PG
Modulation of pKa of active site His-95.

13. Helical Propensities Ala -0.77 Arg -0.68 Lys -0.65 Leu -0.62 Met -0.50
Trp -0.45
Phe -0.41
Ser -0.35
Gln -0.33
Glu -0.27
Cys -0.23
Ile -0.23
Tyr -0.17
Asp -0.15
Val -0.14
Thr -0.11
Asn -0.07
His -0.06
Gly 0
Pro ~3

14. Common Secondary Structure Elements
The Beta Sheet
Take Home Message – Beta sheet is a secondary structural element that is often observed in proteins, description of beta sheet structure – antiparallel strands, hydrogen bonding across strands

15. Secondary structure: reverse turns

16. Secondary Structure: Phi & Psi Angles Defined
Rotational constraints emerge from interactions with bulky groups (ie. side chains).
Phi & Psi angles define the secondary structure adopted by a protein.
Take Home Message: Phi & Psi determine secondary structure
In order to compute secondary structure elements in proteins, you need to understand the spacial constraints that protein sequences are under due to the rotation around peptide bond.

17. The dihedral angles at Ca atom of every residue provide polypeptides requisite conformational diversity, whereby the polypeptide chain can fold into a globular shape

18. Ramachandran Plot
Take Home Message: Ramachradan was a really smart interesting scientist 1922 – 2001
Defined - A graphical representation in which the dihedral angle of rotation about the alpha-carbon to carbonyl-carbon bond in polypeptides is plotted against the dihedral angle of rotation about the alpha-carbon to nitrogen bond.
Ramachandran – calculations that elegantly accounted for the structure and amino acid composition of collagen in 1954, training in physics and electrical engineering – really significant contribution early on to the field of structural biology – developed the above Rhamachandran plot in response to stereochemical critisicisms of the collagen structure – notable critic Crick.
G N Ramachandran used computer models of small polypeptides to systematically vary phi and psi with the objective of finding stable conformations. For each conformation, the structure was examined for close contacts between atoms. Atoms were treated as hard spheres with dimensions corresponding to their van der Waals radii. Therefore, phi and psi angles which cause spheres to collide correspond to sterically disallowed conformations of the polypeptide backbone.
In the diagram above the white areas correspond to conformations where atoms in the polypeptide come closer than the sum of their van der Waals radi. These regions are sterically disallowed for all amino acids except glycine which is unique in that it lacks a side chain. The red regions correspond to conformations where there are no steric clashes, ie these are the allowed regions namely the alpha-helical and beta-sheet conformations. The yellow areas show the allowed regions if slightly shorter van der Waals radi are used in the calculation, ie the atoms are allowed to come a little closer together. This brings out an additional region which corresponds to the left-handed alpha-helix.
Today, for beginners in biochemistry protein structures are introduced with a discussion of the Ramachandran map, which also forms the cornerstone for many discussions of protein folding.

19. Secondary Structure
Table 10

20. Beyond Secondary Structure
Supersecondary structure (motifs): small, discrete, commonly observed aggregates of secondary structures
b sheet
helix-loop-helix
bab
Domains: independent units of structure
b barrel
four-helix bundle
*Domains and motifs sometimes interchanged*

21. Common motifs

22. Supersecondary structure: Crossovers in b-a-b-motifs
Left handed
Right handed

23. EF Hand
Consists of two perpendicular 10 to 12 residue alpha helices with a 12-residue loop region between
Form a single calcium-binding site (helix-loop-helix).
Calcium ions interact with residues contained within the loop region.
Each of the 12 residues in the loop region is important for calcium coordination.
In most EF-hand proteins the residue at position 12 is a glutamate. The glutamate contributes both its side-chain oxygens for calcium coordination.
Calmodulin, recoverin : Regulatory proteins
Calbindin, parvalbumin: Structural proteins

24. EF Fold
Found in Calcium binding proteins such as Calmodulin

25. Helix Turn Helix Motif
Consists of two a helices and a short extended amino acid chain between them. 
Carboxyl-terminal helix fits into the major groove of DNA.  
This motif is found in DNA-binding proteins, including l repressor, tryptophan repressor, catabolite activator protein (CAP)

26. Leucine Zipper

27. Rossman Fold The beta-alpha-beta-alpha-beta subunit
Often present in nucleotide-binding proteins

28. What is a Protein Fold?
Compact, globular folding arrangement of the polypeptide chain
Chain folds to optimise packing of the hydrophobic residues in the interior core of the protein

29. Common folds

30. Tertiary structure examples: All-a
Cytochrome C four-helix bundle
Alamethicin The lone helix
Rop helix-turn-helix

31. Tertiary structure examples: All-b
b sandwich
b barrel

32. Tertiary structure examples: a/b
placental ribonuclease inhibitor a/b horseshoe
triose phosphate isomerase a/b barrel

33. Four helix bundle 24 amino acid peptide with a hydrophobic surface
Assembles into 4 helix bundle through hydrophobic regions
Maintains solubility of membrane proteins

34. Oligonucleotide Binding (OB) fold

35. TIM Barrel The eight-stranded a /b barrel (TIM barrel)
The most common tertiary fold observed in high resolution protein crystal structures
10% of all known enzymes have this domain

36. Zinc Finger Motif

37. Domains are independently folding structural units.
Often, but not necessarily, they are contiguous on the peptide chain.
Often domain boundaries are also intron boundaries.

38. Domain swapping:
Parts of a peptide chain can reach into neighboring structural elements: helices/strands in other domains or whole domains in other subunits.
Domain swapped diphteria toxin:

39. Transmembrane Motifs
Helix bundles Long stretches of apolar amino acids Fold into transmembrane alpha-helices “Positive-inside rule” Cell surface receptors Ion channels Active and passive transporters
Beta-barrel Anti-parallel sheets rolled into cylinder Outer membrane of Gram-negative bacteria Porins (passive, selective diffusion)

40. Quaternary Structure
Refers to the organization of subunits in a protein with multiple subunits
Subunits may be identical or different
Subunits have a defined stoichiometry and arrangement
Subunits held together by weak, noncovalent interactions (hydrophobic, electrostatic)
Associate to form dimers, trimers, tetramers etc. (oligomer)
Typical Kd for two subunits: 10-8 to 10-16M (tight association)
Entropy loss due to association - unfavorable
Entropy gain due to burying of hydrophobic groups - very favourable

41. Structural and functional advantages of quaternary structure
Stability: reduction of surface to volume ratio
Genetic economy and efficiency
Bringing catalytic sites together
Cooperativity (allostery)
There are a number of advantages in forming oligomers:
size without loss of stability
modular construction – one gene – big protein
complex catalytic sites in enzymes
regulation – will return to this briefly later 40

42. Quaternary structure of multidomain proteins