1. Protein Structure 2 Protein ModificationBL

2. Nomenclature
Domain - a particular region within a polypeptide chain (ATP binding domain)
Motif - a protein structural element (may appear more than once in a single protein or in many different types of proteins (e.g. greek key)
Subunit - a single polypeptide unit within a larger multipeptide protein
Complex - group of proteins with long-term or transient physical association

3. Multidomain proteins

5. Multisubunit proteins

6. Quaternary (4°) structure
What are the forces driving quaternary association?
Typical Kd for two subunits: 10-8 to 10-16M!
These values correspond to energies of kJ/mol at 37 C
Entropy loss due to association - unfavorable
Entropy gain due to burying of hydrophobic groups - very favorable!

7. Alcohol dehydrogenase dimer

8. Prealbumin dimer

9. Tyrosine kinases

10. Packing symmetry

11. Multiple subunits vs. multiple domains
Structural factors
Stability: reduction of surface to volume ratio
Bringing catalytic sites together (efficiency)
Flexibility in shared binding sites
Increased complexity
Genetic factors
Gene duplication = genetic freedom/economy
Multiple interactions = increased impact of mutation
Regulation
Cooperativity
Must be co-expressed

12. Tubulin as an example Essential component of the cytoskeleton
Dimers polymerize
Multiple sites of interaction
Dynamic
Mutations have severe effects

13. Protein Modification

14. Post-translational Modifications
Cleavage of signal peptides
Phosphorylation
Amidation
Glycosylation
Hydroxylation
Ubiquitination
Addition of prosthetic groups
Iodination
Adenylation
Sulfonation
Prenylation
Myristoylation
Acylation
Acetylation
Methylation
Oxidative crosslinking
N-Glutamyl cyclization
Carboxylation
Table 4.1

15. Are disulfide bonds post-translational modification?

16. N-terminal modifications
N-formyl Met (by product of translation process and incomplete cleavage by deformylase)
Aminopeptidase cleavage of Met
N-terminal acetylation
N-terminal myristoylation
N-terminal glutamyl cyclization

17. C-terminal modification
C-terminal prenylation
farnesylation
geranylation/geranyl-geranylation
C-terminal amidation

18. Many (but not all) post-translational modifications involve parts of the secretion apparatus

19. Phosphorylation Hydroxyl groups - Ser, Thr, Tyr
Amide groups of His, Lys & Carboxy group Asp
Often causes changes in protein conformation and/or protein-protein interactions

20. C-terminal Amidation Neuropeptides and hormones
C-terminal glycine is hydroxylated alpha-hydroxy-glycine.
Glyoxylate. C-terminal amidation is essential to the biological activity of many neuropeptides and hormones.

21. Hydroxylation and Carboxylation
Hydroxylation (previous lecture)
Lys and Pro (e.g. collagen)
Requires vitamin C
Carboxylation
Requires viatamin K

22. Iodination
Special case: Tyr  thyroxin

23. Protein Acylation Methylation and acetylation S-acylation (Cys)
acetylation of histone Lys
S-acylation (Cys)
Prenylation (C-terminus)
Myristoylation (N-terminus)

24. Acetylation

25. N-terminal acetylation
No simple sequence consensus
Usually accompanied by cleavage of the N-terminal Met

26. Mass spectral analysis

27. Methylation Can occur at carboxyls, amides, sulfhydryl, etc.
SAM is the activated carbon source

28. Multiple methylation

29. Histones DNA binding proteins Chromatin structure
Affect gene expression

30. Sulfonation
O-sulfonation of Ser/Thr discovered in 2004 by MS analysis (Mol Cell Proteomics. 3(5):429-40)

31. N-terminal myristoylation
Consensus Gly-X-X-X-Ser/Thr
myristoyl Coenzyme A

32. C-terminal Prenylation
methylated c-terminus
C-terminal Prenylation
Prenylation
CAAX motif
AAX cleaved and Cys methylated then prenyl group added by thioether linkage (C-S-C).
n-terminus

33. S-acylation

34. Adenylation
Toxins frequently adenylate host target proteins to inactivate them
Glutamine synthetase is inactivated by adenylylation and activated by deadenylylation

35. Adenylation
Adenylation of a DNA ligase Lys residue is a key step in DNA replication and repair

36. All non-cytoplasmic proteins must be translocated
Proteolytic cleavage
All non-cytoplasmic proteins must be translocated
The leader peptide retards the folding of the protein so that molecular chaperone proteins can interact with it and direct its folding
The leader peptide also provides recognition signals for the translocation machinery
A leader peptidase removes the leader sequence when folding and targeting are assured

37. Proteolytic processing
Proteolytic cleavage of the hydrophobic N-terminal signal peptide sequence
Proteolytic cleavage at a site defined by pairs of basic amino acid residues
Proteolytic cleavage at sites designated by single Arg residues
Signal prediction algorithms (e.g. SignalP)
caution: may predict TMDs as signalPs

39. Hormones
All secreted polypeptide hormones are synthesized with a signal sequence (which directs them to secretory granules, then out)
Usually synthesized as inactive preprohormones ("pre-pro" implies at least two processing steps)
Proteolytic processing produces the prohormone and the hormone

40. GASTRIN Product of preprogastrin Signal peptide cleavage
residues
Signal peptide cleavage
prepropeptide  propeptide residues
Cleavage at Lys and Arg residues and C-terminal amidation leaves gastrin
propeptide  peptide
N-terminal residue of gastrin is pyroglutamate
C-terminal amidation involves destruction of Gly

41. GASTRIN

42. GASTRIN
Heptadecapeptide secreted by the antral mucosa of stomach stimulates acid secretion in stomach

43. Glycosylation: 2 flavors
O-linked GalNac
(Ser, Thr, Hyp)
N-linked GlcNAc
(Asn)

44. Glycosylation

45. Protein glycosylation
Usually extracellular or at cell surface
High structural information content
molecular recognition
Occurs along the secretory pathway
Often stabilizes structure
Difficult to get crystal structure for more than one or two carbohydrate residues

46. Protein glycosylation

47. O-linked glycosylation
No consensus sequence for Ser and Thr
Consensus for Hyp
Gly – X – Hyl – Y – Arg
Begins with GalNac transferase (N-acetylgalactosamine)
Mannose common addition to core

48. The ABO(H) blood group determinant is an O-linked polysaccharide
In 1901, Karl Landsteiner discovered blood group antigens.
Since that time nearly 200 antigens have been identified.
Carbohydrate-dependant blood group antigens are carried by both glycoproteins and glycolipids

49. N-linked glycosylation
Most proteins contain several potential sites but usually only a few (in any) are actually glycosylated
Many different patterns
Consensus (-N-X-T/S-)
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50. N-linked glycosylation begins in the cytoplasm
glycosyl-dolichol-phosphate
(lipid) core is transferred to protein

51. Further processing and elaboration occurs in the ER and Golgi

52. Carbohydrates are flexible
Variety of conformations
difficult to get good x-ray data
Structural information
H-bonding
hydrophobic patches
wheat germ agglutinin

53. GPI anchors (glycosylphosphatidyl inositol)

55. Carbohydrates as cross-linkers
Galectin-1 dimer

56. Ubiquitination
DEGRADATION

57. Ubiquitination Ubiquitinating enzymes E1,E2, E3 - thiol ester bond
Final target - isopeptide bond between a lysine residue of the substrate (or the N terminus of the substrate) and ubiquitin
Ubiquitin first activated by adenylation
Science : target protein ubiquitination on Cys!

58. More about proteoglycans and ubiquitination later