Protein Folding in the Cell 1

BIOC 212 Winter 2013 Jason C. Young

Lecture Topics
Protein Folding 1. Elements of protein structure 2. Molecular chaperones 3. Multi-chaperone systems 4. Modifications and Degradation Membranes 1. Membrane lipids 2. Membrane proteins 3. Targeting to endoplasmic reticulum (ER) 4. Sorting and degradation in ER Intracellular Traffic 1. Vesicle formation 2. Vesicle targeting and fusion 3. Lysosome and Nucleus 4. Mitochondria

I am a protein. All living organisms need me to function. A basic building block of the human body, Im made from amino acids found in ribosomes. Proteins give energy to everything from flowers and butterflies to heroes who turn in Communists. I...am a protein. Jack Donaghy, 30 Rock Chain Reaction of Mental Anguish

The Cell

David Goodsell, Scripps

Cellular Proteins
Proteins are the main functional components in cells Genes and mRNA are linear Proteins are made as linear polypeptides by cytosolic ribosomes, but fold into 3-dimensional conformations Folding provides physical stability and functional surfaces The sequence of a protein determines its structure, function and localization

Amino Acids
20 different amino acids Side chains have different chemical characteristics: hydrophobic, polar or charged (acidic or basic) small or large covalently linked into polypeptides

Polypeptides
Peptide bonds in the backbone are uncharged but polar Charge and hydrophobicity of a polypeptide is determined by the side chains Both side chains and backbone can form non-covalent contacts with other amino acids

Polypeptide Backbone
The peptide bond is planar and cannot rotate Rotation around the bonds to the central carbon (C) is possible Therefore, the polypeptide backbone has limited freedom of rotation Some rotation angles between amino acids (residues) in a polypeptide are preferred

Non-Covalent Bonds
Interactions between residues of a polypeptide stabilize structure hydrophobic interactions (exclusion of water) hydrogen bonds van der Waals interactions (transient dipoles between all atoms) ionic bonds

Protein Folding
Folding is driven by hydrophobic interactions other non-covalent interactions and rigidity constraints contribute to the structure Polar side chains usually form outer surface Native State is the most stable conformation Proteins with similar sequences usually have similar native states, and may have similar functions

native state

Disulfide Bonds
Secretory proteins often have covalent disulfide bonds between cysteine side chains extracellular proteins, inside secretory organelles disulfides reinforce structure Cytosolic proteins do not have disulfide bonds cytosol, nucleus, mitochondria

Importance for Folding

hydrophobic interactions hydrogen bonds Van der Waals interactions ionic bonds disulfide bonds very many, strong many, strong many, weak few, strong few, very strong

strong hydrophobic interactions are important for protein structure, and also for membrane formation

Units of Protein Structure

Primary structure amino acid sequence Secondary structure local conformation patterns stretches of polypeptide can have regular arrangements of the polypeptide backbone and position of side chains common structures are -helices and -sheets loops have no regular secondary structure and can be flexible

Alpha-Helix
-helix: backbone is coiled hydrogen bonds between each turn of helix backbone side chains point outwards

Beta-Sheets
-strands backbone is extended almost straight several strands pack sideways into a -sheet hydrogen bonds between the backbone strands side chains on alternate sides

Tertiary Structure
Tertiary structure complete three-dimensional arrangement of the polypeptide secondary structure elements are packed against each other to form the tertiary structure hydrophobic contacts between secondary elements long-range contacts between residues that are far apart in the primary sequence

loops

Units of Protein Structure 4

Quaternary structure: the assembly of multiple polypeptides (subunits) into a final protein interactions between subunits are very stable monomer: single polypeptide with no quaternary structure dimer: two polypeptide subunits trimer, tetramer, 5-mer, 6-mer etc. oligomer: many subunits

haemoglobin tetramer

Domains
A domain is an independently folded unit within a protein Proteins can have one or multiple domains Different domains in a protein often have different functions

Hsp70 ATPase domain

ribbon diagram (polypeptide backbone only)

space-filling model (all atoms)

Polypeptide Length
Most polypeptides are 100 to 800 amino acids long, or from 12 kDa to 90 kDa molecular weight Domains are usually 50 to 200 amino acids long Long proteins have multiple domains
600

500

number of proteins

400

300

200

100

proteins encoded by human chromosome 1

0 < 100 100- 200- 300- 400- 500- 600200 300 400 500 600 700 700- 800- 900- 1000- 1200- 1500- > 800 900 1000 1200 1500 2000 2000

length (amino acids)

Protein Surface
The sequence of a protein determines its functional surface and interactions with ligands Specificity of binding can be narrow (few molecules recognized) or broad (many different molecules) Catalysis: some proteins are enzymes which increase rate of covalent chemical reactions Allostery: conformational changes can change binding surface

Modular Domains
Some types of domains are found in many different proteins Many modular domains form reversible, non-covalent contacts with specific features on other molecules other proteins (different from quaternary structure) certain lipids or carbohydrates allow regulation of function

examples of proteins with modular J domains (green oval)

Some amino acid side chains are chemically similar to each other

Protein Families
Proteins or domains in a family have similar sequences and structures; they usually have related functional mechanisms Similarity (homology) indicates evolutionary conservation Divergent sequences have no similarity and different structures, but may have still have related functions
human Hsp70 ATPase domain E. coli Hsp70 ATPase domain E. coli arsenite transporter ATPase subunit

homologous

not homologous

Which amino acid side chains can form these interactions?

hydrophobic interactions hydrogen bonds Van der Waals interactions ionic bonds disulfide bonds

can the polypeptide backbone form any of these interactions?

End of 1
Molecular Biology of the Cell, Alberts et al. 4th or 5th Ed. Ch. 3, protein structure, protein function Dobson (2003) Protein Folding and Misfolding. Nature 426, 884890.