Protein Structure and Organization

Protein Structure

• Proteins have 4 levels of organization: primary, secondary, tertiary, and quaternary
• Primary structure: specific sequence of amino acids, with 20 possible amino acids, resulting in 20^n variations
• Side chain (R group) is the portion of each amino acid that gives it unique properties
• Polypeptide backbone with peptide bonds and side chains
• Amino terminus (N-terminus) and carboxyl terminus (C-terminus) are the ends of the protein
• Sequence is determined by the sequence of the gene encoding the protein (DNA)

Secondary Structure

• Folding patterns: alpha helix and beta pleated sheets
• Alpha helix: hydrogen bond between carbonyl of one amino acid and N-H of another, every 4th amino acid
• R groups project outwards, can be left-handed or right-handed
• Beta pleated sheets: hydrogen bonds extend from one part of the chain to another, with amino acids interacting with adjacent lines
• R groups are exposed, pointing up or down, with arrows pointing from N to C
• Coiled coil: two alpha helices wrapped around each other, mediated by hydrophobic residues

Tertiary Structure

• Native conformation, mediated by hydrophobic interactions (hydrophobic core)
• Interactions between R-groups: van der Waals forces, hydrogen bonds, ionic bonds, hydrophobic interactions, and disulfide bridges
• Two types: fibrous and globular
• Globular proteins are more compact, with polypeptide chains folding into complex shapes
• Fibrous proteins have an elongated shape, forming long strands or flattened sheets

Quaternary Structure

• Linking of multiple proteins to form large complexes with multiple subunits
• Some proteins stop at tertiary structure, while others form quaternary structures
• Rely on intermolecular interactions of R groups (disulfide bonds, noncovalent bonds)
• Homo: same subunits, hetero: two or more different subunits

Protein Aggregates

• Can cause disease, such as neurodegenerative diseases
• Structure goes from monomer to dimer to oligomer to aggregate
• Not sure what causes aggregates to form, but oxidative stress may be a factor
• Aggregates tend to form later in life, and can be a symptom of neurodegenerative diseases

Protein Function

• Protein-protein interactions can be stable and involved in important cell functions
• Interacting proteins typically have complementary surfaces
• Protein functions can be figured out based on the types of proteins it interacts with
• Protein families: group of proteins that share origin, related function or similar sequence/structure

Post-Translational Modification

• Addition or removal of molecules can regulate protein function
• Addition of phosphates can increase or decrease protein activity
• Kinase: enzyme that adds phosphate, phosphatase: removes phosphate
• Signalling cascades: reactions that occur in response to a stimulus, controlled by phosphorylation and dephosphorylation

Other Protein Techniques

• Fluorescence microscopy: uses proteins with fluorescent tags or labels
• FRET (Forster Resonance Energy Transfer): visualizes protein interactions
• FRAP (Fluorescence Recovery After Photobleaching): studies protein movement
• Isolating specific proteins from within cells: protein purification, cell fractionation, and column chromatography

Organelles

• Anything in the cell with a membrane is an organelle
• Most organelles overlap between plant and animal cells
• Cytoplasm: everything except the nucleus, cytosol: everything outside membrane-bound organelles
• Mitochondria and chloroplasts: ATP production
• ER: most membrane protein and soluble organelle protein synthesis
• Golgi apparatus: sorting of proteins and lipids from ER
• Nucleus: contains genome, DNA and RNA synthesis
• Endosomes: move material between organelles and uptake extracellular molecules
• Lysosomes: degradation of unneeded intracellular organelles and extracellular molecules### Protein Synthesis and Secretory Pathway
• Proteins that enter the secretory pathway must first enter the Endoplasmic Reticulum (ER)
• Proteins have hydrophobic stop sequences to stay in the membrane
• Orientation of proteins in the ER membrane is important for their function in the plasma membrane

Membrane Proteins

• Membrane proteins have multiple hydrophobic stop sequences to stay in the membrane
• Proteins can be linked to the membrane with a Glycosylphosphatidylinositol (GPI) anchor, replacing hydrophobic stop sequences
• GPI anchor is used when the sequence needs to be cleaved to function but still needs to be in the membrane

Protein Translation

• Protein translation in the cytosol occurs on "free" ribosomes
• Proteins that finish synthesis in the cytosol (cytosolic proteins) will never enter the endomembrane system unless needed for an organelle or to leave the cell

Folding and Modification in the ER

• ER proteins are folded by chaperones like BiP, GRP94, Calnexin (CNX), and Calreticulin (CRT)
• PDI (protein disulfide isomerase) helps form disulfide bridges correctly in the ER
• ER protein modification: enzymes modify proteins, commonly by glycosylation (addition of sugars onto proteins)
• Glycosylated proteins are known as glycoproteins
• Glycosylation helps proteins fold and aids interaction with other molecules
• Nearly all proteins in the ER are modified co-translationally by glycosylation

Quality Control

• If proteins don't fold properly, they can interfere with other proteins, aggregate, and activate wrong signals
• Strict quality control: improperly folded proteins are sent to the cytosol to be degraded by the proteasome
• Time is allowed for proteins to try to fold properly
• Glycosylation allows for this quality control: sugars get added to the protein, and then two are trimmed by glucosides

Unfolded Protein Response (UPR)

• If large amounts of unfolded/misfolded proteins accumulate in the ER, the UPR is triggered
• More chaperones and proteases that degrade defective proteins are made
• Synthesis of new proteins not involved in the UPR is stopped

Vesicle Transport

• Budding vesicles have a 'coat' of soluble cytosolic proteins that assemble on the membranes of the donor compartment
• COP I transports proteins backwards from the Golgi to the ER and within the Golgi
• COP II transports proteins forward from the ER to the Golgi
• Clathrin-coated vesicles sort proteins to lysosomes and vacuoles
• COP II coat is a large complex that includes SAR1, a GTP-bound protein

Golgi Apparatus

• Located next to the nucleus, but not connected (ER is connected to the nucleus through the perinuclear compartment)
• Consists of stacks of membranous cisternae (ribbon-like)
• Functionally distinct compartments (each ribbon does something different)
• The Golgi is a "processing plant" that takes proteins from the ER and processes, modifies, and sorts them

Protein Modification in the Golgi

• Proteins are processed (like insulin cleavage), modified (like glycans), and sorted in the Golgi
• Proteins move from the ER to the cis-Golgi, then to the medial Golgi, and finally to the trans Golgi
• Proteins are sorted to determine which ones need to go back to the ER vs which will move through the Golgi
• Two hypotheses for maintaining polarized Golgi structure: vesicle transport mechanism and cisternal maturation mechanism

Golgi Exocytosis

• Contents of secretory vesicles are released into the extracellular environment
• Vesicle lumen becomes part of the extracellular space
• Soluble proteins are released, and membrane proteins get inserted into the plasma membrane
• Glycosylation sites are on the outside of the cell

Regulated Secretion

• Constitutive secretion: synthesis and secretion of proteins continuously in an unregulated manner (most membrane proteins and extracellular matrix proteins)
• Regulated secretion: proteins to be secreted are densely packaged into large vesicles (secretory granules) that wait for a cellular signal before releasing their contents (hormones, neurotransmitters, digestive enzymes)

Lysosome Sorting

• Sorting of proteins to lysosomes uses mannose 6 phosphate (m6p)
• m6p can bind to an m6p receptor at non-acidic pH
• In the lysosome, the m6p and m6p receptor dissolve apart