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

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Protein Structure and Function

Electron micrograph of insect flight tissue

In cross section shows an array of 2 protein filaments

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DNA polymerase III – DNA complex (Replication)

Structure and Flexibility indicates Function

Conformational change of lactoferrin upon binding of Fe

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Proteins are Polypeptides

Direction of a Protein

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Cys can cross-link between 2 polypeptide chains -> Disulfide bridge

Covalent cross-link on 3° structure level

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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

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Examples of α-Helical Proteins:

Many membran proteins are α-helical

Bacteriorhodopsin (Photoreceptor)

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Examples of β-sheet Proteins:

Fatty acid binding protein -> β barrels structure

AntibodiesOmpX: E. coli porin

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Quaternary Structure:

Polypeptide chains assemble into multisubunit structures

Cell-surface receptor CD4

Cro protein phage λ

Tetramer of hemoglobin Coat protein of rhinovirus

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Protein Folding

Folding is a highly cooperative process (all or none)

Folding by stabilization of Intermediates

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Protein Folding by Chaperons

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Protein Modifications

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Protein Modifications

GFP fluorescent: Rearrangement and oxidation of Ser-Tyr-Gly

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Function of Proteins

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Protein Trafficking

Bovine cell stained with fluorescent dyes.

Green -> ER

Red -> Mitochondria

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Major Protein sorting pathways in Eukaryotes

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Secretory proteins are transported to ER shortly after synthesis started

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

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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

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N-terminal signal sequence of secretary and membrane proteins

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Sec61α is a translocon component

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Post-translational Translocation

Fairly common in yeast and occationally in higher eukaryotes.

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Integral Membrane Proteins synthesized in ER

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Synthesis and insertion into the ER of membrane proteins

Type I

Type II

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GPI-anchored Proteins

Glycosylphosphatidylinositol (GPI) From yeastIn other organisms -> differs in1.Acyl chain2.Carbohydrate moiety

Formation of GPI-anchored proteins in the ER membrane

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Hydropathy profiles to identify topogenic sequences

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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

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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

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Addition of N-linked sugars in the ER

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Processing of N-linked glycoproteins in the Golgi apparatus of mammalien cells

Mannose trimming

Gucosamine addition

Galactose addition + neuraminic acid linkage to galactose

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

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

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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

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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

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Modification of Proteins - Proteolytic Cleavage

Proteolytic cleavage of proinsulin occurs in secretory vesicles (after Golgi)

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Transport of proteins to other organelles

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Export of Bacterial Proteins

Post-translational translocation across inner membrane of gram-negative bacteria

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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

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The secretory and endocytic pathway of protein sorting

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Protein Transport between Organelles are done by Vesicles

Assembly of protein coat drives vesicle formation and selection of cargo molecules

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Assembly and Disassembly of Coat protein

Interaction of cargo protein with vesicleN-terminus of Sar1 (membrane anchor) not shown

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Model for Docking and Fusion of Transport vesicles with Target Membrane

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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

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

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Clathrin Coats

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Receptor-Mediated Endocytosis

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Receptor-Mediated Endocytosis

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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

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HIV Budding from Plasma Membrane

Gag, ESCRT and Vps4 proteins are neededESCRT lacking -> no budding (accumulation of incomplete viral buds on membrane