1. Introduction to Protein StructureQ:
Whats that? A:
Something, you get Noble prize for...
John Kendrew & Max Perutz 1962
Structures of myoglobin & hemoglobin

2. Subjects, covered in this lecture
Amino acids and their properties
Peptide geometry
Secondary structure
Motifs
Domains
Quaternary structure

3. Why bother about protein structure?
Gives you an visual image of how proteins look like.
Study of protein structures allows to gain an insight into how protein really accomplish their function.
Nobel prizes...

4. Amino Acids
20 different ones, sharing a common backbone but varying side chain.
Classed according to their chemical properties
L-form

5. nonpolar amino acids R group consists of carbon chains
leucine and isoleucine
are structural isomers

6. nonpolar amino acids - R group consists of carbon chains
Phenylalanine and tryptophan
have aromatic rings which are flat due to the double bond network
Tryptophan is often classified as being polar because of the NH group. In practice, however it has more of hydrophobic properties
Proline has its R group bound to the amino nitrogen to form a ring network
Methionine has a sulphur atom in its sidechain
sulphur has the same valence as oxygen

7. polar amino acids
- R group consists of carbon, oxygen and nitrogen atoms
together they make the sidechain more hydrophilic
Ser and thr are a mix of carbon chains and hydroxyl functional groups (-OH). Cysteine has a thiol group (-SH) which is otherwise structurally similar to serine
but not chemically similar
Asn and gln have an
amide functional group

8. charged amino acids
- R group has a charge at physiological pH (7.4). pK of the charged groups vary
carboxyl
group
carboxyl
group
amino
group
guanidinio
group
imidazole
group, sometimes charged
Most often classified as a polar amino acid

9. Cysteine and disulphides
The almost exclusively only way to covalently link two non-sequential residues is by forming a disulphide bridge
Formation of disulphide requires an oxidative environment, threfore disulphides are very rare in intracellular proteins but quite abundant in secretory proteins

10. Peptide units A peptide is a set of covalently bonded amino acids.
The covalent bond is usually referred as peptide bond

11. Structural biologist’s
peptide unit – from Ca to next Ca - all main chain atoms within the unit lie in a plane
Biochemist’s peptide unit –from N to C – all main chain atoms within the unit lie in the same residue

12. Angles phi (f) N-Ca
Angle psi (y) Ca-CO
Angle omega (w) C-N

13. The w angle, cis- and trans- peptides
Because of the partly double nature of peptide bond, w is always close to 180o for trans- peptides or 0o for cis- peptides (±30o in exterme cases)
Cis- peptides are energetically extremely unfavourable (~1000 fold) because of steric clashes between the neighbouring Ca atoms

14. The only exception is peptide bond before proline, where cis- peptide is just 4 times less favourable than trans- peptide, because there are some steric clashes in both cis- and trans- forms
Proline cis-trans isomerization is an important factor in protein folding, which is why there are special enzymes – prolylpeptydyl isomerases to catalyze the transition from one form to another

15. According to statistics, 0. 03% of non-proline peptides and 5
According to statistics, 0.03% of non-proline peptides and 5.2% of X-Pro peptides are in cis- conformation, resulting in a total of 0.3 % cis-peptides
In most cases cis- peptides, especially non-proline, occur for a good reason, for example to maintain some particular conformation in the active site of enzyme

16. Main chain conformations (I)
Only certain combinations of y and f are allowed, due to steric clashes of backbone atoms and Cb atom. Plot of these combinations yields the Ramachandran plot.
All amino acids clusters in specific regions (called allowed regions) except Gly (explains why Glycine is an important amino acid).

18. In good quality structures only about 2% of amino acid residues are found in the disallowed regions of Ramachandran plot
Of course, residues with disallowed conformations often have some important function in proteins

19. Side chain conformations
Side chains can have in principle different conformations (rotation of Ca-Cb...)
The observed conformations in protein structures are the ones which are more energetically favourable (rotamers).

20. Name three amino acids which are very different from others!
Proline
No free amino group
Very rigid
Introduces breaks in a helices and b strands
Glycine
Lacks a side chain
Can be found anywhere in Ramachandran plot
In proteins often found in flexible regions with unusual backbone conformations
Cysteine
Disulphides

21. Primary, secondary, tertiary and quaternary structures

22. The hydrophobic core
The hydrophobic sidechains of protein has a tendency to cluster together in order to avoid unfavourable contacts with polar water molecules
As a result, in general, hydrophobic sidechains are located in the interior of protein, forming the hydrophobic core
Polar and charged amino acids usually are located on the surface of the protein
Polar and charged residues also can make hydrophobic contacts with their aliphatic carbon atoms
Polar and charged residues are seldom completely buried within the core and even when they are, the polar groups are almost invariably involved in hydrogen bond formation

23. The reasons of secondary structure formation
Since sidechains of hydrophobic residues are located in the hydrophobic core, the mainchain atoms of the same residues in most cases are also within the hydrophobic core
Since the presence of polar groups in hydrophobic environment is very unfavourable, the main chain N- and O- atoms have to be neutralised by formation of hydrogen bonds
The two most efficient ways of hydrogen bond formation is to build an alpha helix or a beta sheet

24. The alpha helix
3.6 residues per turn
the hydrogen bonds are made between residues n and n+4

26. Variants of alpha helix
In regular a helix, residue n makes a H-bond with residue n+4
In 310 helix, residue n makes a H-bond with residue n+3. There are 3 residues per turn, connected by 10 atoms, hence the name 310
In p helix, residue n makes a H-bond with residue n+5
In p helix there is a hole left in the middle of helix and in 310 helix the main chain atoms are packed very tightly. None of above is energetically favourable
310 and especially p helices occur rarely and usually only at the ends of regular a helix or as a separate single-turn helix

27. Handedness of alpha helix
The a helices as well as 310 and p helices ale almost exclusively right-handed
In very rare occasions, left handed a and 310 helixes can occur. They are always very short (4- 6 residues) and normally involved in some important function of protein like in active site or ligand binding
There are about 30 reported cases of left-handed helices. In contrast, the number of known right handed helices is of order of hundreds of thousands

28. The dipole moment of a helix

29. Good and bad helix formers
Different side chains have been found to have weak but definite preferences for helix forming ability
Ala, Glu, Leu and Met are good helix formers
Pro, Gly, Tyr and Ser are very poor helix formers
The above preferences are not strong enough to be used in accurate secondary structure predictions

30. Periodic patterns in a helices
The most common location of an a helice is along the outside of protein, with one side of the helix facing the hydrophobic core and other side facing the solvent
Such a location results in a periodic pattern of alterating hydrophobic and polar residues
On itself, however, the pattern is not reliable enough for structure prediction, since small hydrophobic residues can face the solvent and some helices are completely buried or completely exposed

31. Beta sheets
Antiparallel
Parallel

32. A mixed b sheet
A mixed b sheet is far less common than antiparallel or parallel

33. Twist in b sheets
Almost all b sheets in the known protein structures are twisted
The twist is always right-handed

34. Loops Loops connect secondary structure elements
Loops are located on the surface of protein
In general, main chain nitrogen and carbonyl oxygen atoms do not make H-bonds each to other in loops
Loops are rich in polar and charged residues
The lenght of loops can vary from 2 to more than 20 residues
Loops are very flexible, which makes them difficult to see in either x-ray or NMR studies of proteins
Loops frequently participate in forming of ligand binding sites and enzyme active sites
In homologous protein families loop regions are far less conserved than secondary stucture elements
Insertions and deletions in homologous protein families occur almost exclusively in loop regions

35. Hairpin loops and reverse turns
Loops, which connect two adjacent antiparallel beta strands are called hairpin loops
2 residues long hairpin loops are often called reverse turns, beta turns or simply turns
Type I turn Type II turn
Hairpin loop
Strand1
Strand2

36. Motifs
Simple combinations of a few secondary structure elements occur frequently in protein structures
These units are called supersecondary structure or motifs
Some motifs can be associated with a specific biological function (e.g. DNA binding)
Other motifs have no specific biological function alone, but are part of larger structural and functional assemblies

37. Helix-loop-helix motifs
DNA binding motif
Calcium binding motif

38. The hairpin b motif
Two adjacent anti-parallel b strands, joined by a loop
The hairpin motif can occur both as an isolated unit or as a part of bigger b sheet
Snake venom- erabutoxin
Bovine trypsin inhibitor

39. 24 different ways to connect two b hairpins
Only the first 8 arrangements exist in known proteins

40. The Greek key motif
The most common way to connect 4 adjacent antiparallel b strands
The Greek key motif in Staphilococcus nuclease

41. The b-a-b motif A convinient way to connect two paralel beta strands
b-a-b motif is a part of almost all proteins, containing a paralel beta sheet

42. The handedness of b-a-b motif
Theoretically, two distinct “hands” can exist in b-a-b motif, with a helice above or below the plane of beta sheet
In almost all cases the right handed motif exists R L

43. Domains
Domain ia a polypeptide chain or a part of a polypeptide chain that can fold indepedently in a stable tertiary structure with its own hydrophobic core
Domains can be formed from several simple motifs and additional secondary structure elements
Proteins can have anything from one to several tens of domains
In proteins with sevaral domains, most often each domain is associated with a distinct biological function

44. 2xb hairpin + b strand
16xb-a-b
hsh-rnase inhibitor
2x Greek key

45. Domains are most often, but not always continuous pieces of primary structure

46. Example of proteins with several domains - lac repressor
Helix-turn-helix domain (binds to DNA)
hinge helix
Core domain, containing two subdomains, which in turn contain several b-a-b motifs (binds ligand)
C-terminal helix (tetramerization)

47. Intact IgG contains 12 immunoglobulin-like domains
Each domain is made of two beta sheets with a topology similar to two Greek key motifs

48. The quaternary structure
Some proteins are biologically active as monomers. For those proteins quaternary structure does not exist
Other proteins, however, are active as homo- or hetero- polymers
The simplest case and by far the most common form of quaternary structure is a homodimer
The monomers in homopolymers are often arranged in a symmetric fashion with one or several symmetry axes going through the molecule or some sort of helical arrangement
Some biologically active units have a very complicated quaternary structure –like ribosomes or viral capsids

49. 2-fold symmetry in Glutahione-S-transferase

50. 9-fold symmetry in light-harvesting complex II from Rhodopseudomonas acidophila.

51. 222 symmetry in prealbumin

52. A simple icosahedral virus – 180 chemically identical subunits

53. Small subunit of ribosome: a lot of different proteins, no symmetry