1. Protein Structure and Function
Electron micrograph of insect flight tissue
In cross section shows an array of 2 protein filaments

2. Structure and Flexibility indicates Function
DNA polymerase III – DNA complex (Replication)
Conformational change of lactoferrin upon binding of Fe

4. Proteins are Polypeptides
Direction of a Protein

5. Cys can cross-link between 2 polypeptide chains -> Disulfide bridge
Covalent cross-link on 3° structure level

6. Examples of α-Helical Proteins:
α-helical coiled coil proteins:
Form superhelix
Found in myosin, tropomyosin (muscle), fibrin (blood clots), keratin (hair)
The cytoskeleton is rich in filaments which are α-helical coiled coil proteins

7. Examples of α-Helical Proteins:
Bacteriorhodopsin (Photoreceptor)
Many membran proteins are α-helical

8. Examples of β-sheet Proteins:
Fatty acid binding protein -> β barrels structure
OmpX: E. coli porin
Antibodies

9. Quaternary Structure:
Polypeptide chains assemble into multisubunit structures
Cell-surface receptor CD4
Cro protein phage λ
Tetramer of hemoglobin
Coat protein of rhinovirus

10. Protein Folding Folding is a highly cooperative process (all or none)
Folding by stabilization of Intermediates

11. Protein Folding by Chaperons

12. Protein Modifications

13. Protein Modifications
GFP fluorescent: Rearrangement and oxidation of Ser-Tyr-Gly

14. Function of Proteins

15. Protein Trafficking Bovine cell stained with fluorescent dyes.
Green -> ER
Red -> Mitochondria

16. Major Protein sorting pathways in Eukaryotes

17. Secretory proteins are transported to ER shortly after synthesis started

18. Synthesis of secretory proteins and their cotranslational translocation across the ER membrane

19. Synthesis of secretory proteins and their cotranslational translocation across the ER membrane
What is needed for translocation:
Signal sequence (9-12 hydrophobic AA with some mainly pos. charged ones – in some prokaryotes sometimes longer, most of the times cleaved off by peptidases on the ER lumen side, sequence mainly at N-terminal)
Signal-Recognition-Particle (SRP) –recognizes signal sequence of ribosome complex (ribosome with mRNA), redirects ribosome complex to SRP receptor, puts synthesis of protein on hold
SRP receptor – binds the ribosome- SRP complex - driggers that ribosome complex is moved to translocon (GTP dependent)
Translocon is a protein channel, opens upon binding of ribosome complex, synthesis through channel

20. N-terminal signal sequence of secretary and membrane proteins

21. Sec61α is a translocon component

22. Post-translational Translocation
Fairly common in yeast and occationally in higher eukaryotes.

23. Integral Membrane Proteins synthesized in ER

24. Synthesis and insertion into the ER of membrane proteins
Type I
Type II

25. GPI-anchored Proteins
Glycosylphosphatidylinositol (GPI)
From yeast
In other organisms -> differs in
Acyl chain
Carbohydrate moiety
Formation of GPI-anchored proteins in the ER membrane

26. Hydropathy profiles to identify topogenic sequences

27. Protein Modification
Membrane and soluble secretary proteins synthesized on the ER have 4 possible modifications before the reach final destination:
Glycosylation in ER and Golgi
Formation of S-S bonds in ER
Proper folding and assembly of multisubunits in ER
Proteolytic cleavage in ER, Golgi, and secretory vesicles

28. Protein Modification - Glycosylation
O-linked glycosylkation:
Attachment of sugars to OH of Ser and Thr
Often contain only 1-4 sugar groups
N-linked glycosylation:
Attachment of sugars to amine N of Asn (Asn-X-Ser/Thr)
Larger and more sugar groups -> more complex
Glycosylation patters differ slightly between spieces !!!
In Yeast:
N-linked glycosylation are classified as core and mannan types. The core type contains mannoses whereas the mannan-type structure consists of an inner core extended with an outer chain of up to mannoses, a process known as hyperglycosylation.
Precursor of N-linked sugars that are added to proteins in the ER

29. Addition of N-linked sugars in the ER

30. Processing of N-linked glycoproteins in the Golgi apparatus of mammalien cells
Galactose addition + neuraminic acid linkage to galactose
Gucosamine addition
Mannose trimming

31. Formation of S-S bond by Protein Disulfide Isomerase (PDI)

32. Pathways for formation of S-S bonds in Eukaryotes and Bacteria

33. Folding and assembly of Multimers
Hemagglutinin trimer folding
Binding of Chaperone BiP
Closing S-S bond, N-linked glycosylation
Membrane anchoring
Assembly of trimer
Another example for assembly of multimers -> immunoglobulins

34. Improperly Folded Protein Induce Expression of Chaperons
Unfolded and incomplete folded protein in the ER
-> releases chaperons (BiP) from Ire1
-> upon release of BiP Ire1 dimerizes (activation) -> Endonuclease activity in th cytosol -> splices Transcription factor Hac1 -> Hac1 protein returns into nucleus -> activates transcription of Chaperons
-> Misfolded and unassembled proteins -> transported from the ER to the cytosol -> degradation

35. Modification of Proteins - Proteolytic Cleavage
Proteolytic cleavage of proinsulin occurs in secretory vesicles (after Golgi)

36. Transport of proteins to other organelles

37. Export of Bacterial Proteins
Post-translational translocation across inner membrane of gram-negative bacteria

38. Injection of Protein by Pathogenic Bacteria (into Animal cells)
Yersinia pestis:
Causes Pest
Virulence: Disables host macrophages
-> by injecting a small set of proteins into macrophages
Secretion mechanism for injecting bacterial proteins into Eukaryotic cells

39. The secretory and endocytic pathway of protein sorting

40. Protein Transport between Organelles are done by Vesicles
Assembly of protein coat drives vesicle formation and selection of cargo molecules

41. Assembly and Disassembly of Coat protein
Interaction of cargo protein with vesicle
N-terminus of Sar1 (membrane anchor) not shown

42. Model for Docking and Fusion of Transport vesicles with Target Membrane

44. Vesicle-mediated Protein Trafficking between ER and Golgi
Backtransport mainly used for:
-> recycling of membrane bilayer
-> recycling of proteins (SNARE)
-> missorted proteins
Normal transport of secretory proteins

45. Involvement of the 3 major types of coat proteins in traffic and secretory pathways

47. Clathrin Coats

48. Receptor-Mediated Endocytosis

49. Receptor-Mediated Endocytosis

50. Membrane Fusion directed by Hemagglutinin (HA)
Influenza Virus:
Glycoprotein on suface of virus
After endocytosis (uptake of virus of the cell) viral envelop fuses with endosomal membrane
Acidic pH necessary for conformational change in HA -> viral HA can insert into endosomal membrane

51. HIV Budding from Plasma Membrane
Gag, ESCRT and Vps4 proteins are needed
ESCRT lacking -> no budding (accumulation of incomplete viral buds on membrane