1

Protein Structure and Function

Electron micrograph of insect flight tissue

In cross section shows an array of 2 protein filaments

2

DNA polymerase III – DNA complex (Replication)

Structure and Flexibility indicates Function

Conformational change of lactoferrin upon binding of Fe

3

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:

Many membran proteins are α-helical

Bacteriorhodopsin (Photoreceptor)

8

Examples of β-sheet Proteins:

Fatty acid binding protein -> β barrels structure

AntibodiesOmpX: E. coli porin

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:

1.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)

2.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

3.SRP receptor – binds the ribosome- SRP complex - driggers that ribosome complex is moved to translocon (GTP dependent)

4.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 yeastIn other organisms -> differs in1.Acyl chain2.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:

1. Glycosylation in ER and Golgi

2. Formation of S-S bonds in ER

3. Proper folding and assembly of multisubunits in ER

4. 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 13-14 mannoses whereas the mannan-type structure consists of an inner core extended with an outer chain of up to 200-300 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

Mannose trimming

Gucosamine addition

Galactose addition + neuraminic acid linkage to galactose

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)

Secretion mechanism for injecting bacterial proteins into Eukaryotic cells

Yersinia pestis:

Causes Pest

Virulence: Disables host macrophages

-> by injecting a small set of proteins into macrophages

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 vesicleN-terminus of Sar1 (membrane anchor) not shown

42

Model for Docking and Fusion of Transport vesicles with Target Membrane

43

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

46

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 neededESCRT lacking -> no budding (accumulation of incomplete viral buds on membrane