module 2

help to prevent inappropriate interactions between amino acid residues and increase the efficiency of protein folding

• Molecular chaperones (monomeric) • Chaperonins (multimeric) -both chaperone types assist protein folding in the same way. They help proteins fold by preventing the formation of incorrect folding within the molecule or the inappropriate associations of proteins with other proteins.

-first identified as proteins that are expressed at high levels under conditions of stress (elevated temp), if cell is exposed to high temp, it responds by making more Hsp to refold various proteins in the cell Hsp70: cytosol and mitochondria (eukaryotic cells) BiP :in endoplasmic reticulum DnaK: in bacteria Hsp bind to hydrophobic R groups and prevent the nascent polypeptide from associating with other proteins or from folding prematurely, and from aggregating with other hydrophobic residues.

Hsp70 contains 2 domains: nucleotide binding domain and substrate binding domain nucleotide binding domain: will bind ATP which will be used as an energy source substrate: will be an unfolded protein that is just leaving the ribosome and has not yet folded How is protein folding assisted? 1. hydrophobic residues on HSP 70 allow it to rapidly bind the hydrophobic patches on unfolded proteins. From there, ATP hydrolysis to ADP changes the confirmation of the HSP 70 chaperone. This changes the relative shape of the target protein, allowing it to fold properly. 2. ATP hydrolysis is stimulated by co chaperone DNA J or HSP 40. 3. ADP is released from HSP 70 (assisted by the nucleotide exchange factor - GrpE or BAG1) 4. A new ATP then comes in to fill the nucleotide binding domain. Once this occurs, the folded protein is released and the HSP70 is ready to bind to another unfolded protein.

Large cylindrical macromolecular assemblies that form an isolation chamber or barrel for newly synthesized polypeptides that allows them to fold without interference from other macromolecules TCiP: eukaryotic cytosol GroEL: bacteria or chloroplast Hsp 60: mitochondria

chaperonin complex consists of 2 large GroEL subunits. Multiple proteins form the walls of the 2 GroEL (large subunits) that are attached to one another at their bases. Each of the tops of the chambers are alternately opened or capped by a GroES (small) subunit - like a lid on the GroEL barrel. protein folding steps (keep in mind that 2 groEL chambers are used in an alternating fashion. only 1 groEL chamber is helping with protein folding at a given time): 1. bottom chamber is releasing the groES cap and ADP as it completes its activity. At the same time, the top chamber is binding to ATP and a new substrate peptide. A new groES cap binds to the top of the groEL, closing the chamber and isolating the substrate polypeptide. 2. A conformational change enlarges the chamber dimensions, giving the target peptide room to fold. Chamber remains closed for several seconds as the protein in isolation is allowed to fold (chaperonin does not fold the protein directly it allows it to fold inside it) 3. ATP hydrolysis then allows the groES cap to come off and the folded protein diffuses out -> if it didn’t fold, it may repeat the entire process but tyje chaperonin will use the bottom chamber instead

• tight (in absense of GroES cap) and relaxed (in association with GroES - inside becomes larges as cap binds, and chamber opening narrows) conformation Conformational changes are observed in GroEL upon association with GroES GroEL shifts to larger “relaxed” conformation, releasing protein within chamber

One subunit of GroEL = Hsp60 • 7 Hsp60 subunits are contained in a chaperonin (each Hsp60 binds to a single ATP molecule) 1 Hsp60 protein has 3 domains: apical, intermediate and equatorial domains

• in order to change shape of groEL chamber, each of the 7 HSP60 subunits has to change shape • there is an open/tight confirmation where each HSP60 protein is bent at a hinge. • there is a relaxed confirmation where each HSP60 protein is bound to an ATP and is in an elongated confirmation (when groEL is closed by groES lid) it is the ind. behaviour of the HSP subunits that leads to overall chaperonin function

unfolded proteins are a risk to the cell as they lack appropriate functions, are likely to aggregate and form complexes that are harmful to the cell, so… Cells must degrade: * misfolded proteins * denatured proteins * proteins at too high a concentration * proteins taken up into the cell (endocytosed into cell) * regulated proteins (cyclically made and degraded during cell cycle) when they need to be degraded, they are tagged and sent to proteasome for degradation

Step 1: Tagging of the protein by covalent attachment of ubiquitin molecules (to specific aa residue within the polypeptide sequence of the target protein) - ubiquitin: seen in all organisms, it is in all sub-cellular compartments where protein folding occurs. Not specific to a subset of proteins, but can assist many diff. proteins (w/distinct structures and functions) * Small protein, 76 residues Step 2. Degradation of the tagged protein into short peptides (7-8 residues) by the proteasome (ubiquitin tag is recognized by the proteolytic machinery, proteasome of the cell, and the target protein is cleaved into short peptide sequences, effectively eliminating protein function.

Addition of ubiquitin to a protein targets that protein for degradation by proteasome. 3 enzyme system: * E1: Ubiquitin activating enzyme (A1 recognizes free ubiquitin protein in the cytosol and picks it up) • E2: Ubiquitin conjugating enzyme (will facilitate attachment of ubiquitin to the target protein) • E3: Ubiquitin Ligase = large family of proteins, each member recognizes a different signal (recognizes the specific target for degradation and attaches ubiquitin to it)

• Ubiquitin activated by linkage to E1 (E1 is attached to ubiquitin. this requires energy from ATP hydrolysis) • Activated ubiquitin is transferred to Cys on E2 (ubiquitin is transferred over to E2) 3.E3 recognizes substrate and transfers ubiquitin to lysine side chain of target substrate (target protein, has been previously recognized by E3. E3 and E2 interact and the ubiquitin is transferred from E2 to the target protein) • Poly-ubiquitinylation (multiple ubiquitins are subsequently added in this step. Once poly-ubiquitination occurs, the protein can now be recognized for degradation. overall, this protein targeting process for degradation is amplified in the cell (E1 combined with many E2’s + hundreds of E3’s), E3 enzyme is the one that can provide the specificity of degradation

degradation occurs within a large protein complex called a proteasome (similar to chaperonin, wall is created from identical subunits to create a hollow interior barrel with caps at the ends) • Core forms a central hollow cylinder with proteolytic activity (function: diff. from chaperonins, central cylinder contains proteolytic enzymes that will break down any protein found within this core) • Caps on each end form narrow openings through which unfolded polypeptides are threaded

steps: - poly-ubiquitin tag is recognized by the cap of the proteasome. from there, target protein is unfolded as it enters into narrow opening of the cap - ubiquitins are removed prior to proteasome entry - since ubiquitin proteins are not degraded, they can be used again - as the target protein enters the proteasome core, it is cleaved by the non specific proteolytic activity of the complex - end result: small peptides leave at the other end of the proteasome. These can be further degraded by cytosolic proteases or within the lysosome of the cell. The polyubiquitinated protein is unfolded as it is threaded into the proteasome. * The polypeptide is cleaved into small peptides (2-24 aa) which are released and further degraded.

spinocerebeller ataxia (neurodegenerative disease): a mutation in the ataxin-1 gene creates a misfolded ataxin protein. That misfolded protein is still tagged w/ubiquitin and carried to the proteasome but it cannot be unfolded. Instead, it gets stuck on the proteasome and as a result, the ataxin protein builds up in the cell, forming lethal aggregates. additional problem: these misfolded proteins can prevent proteasome function by saturating the netry sites and preventing the appropriate degradation of many other proteins in the cell.

they must interact with other molecules

molecule in which the protein interacts with, molecule that is bound by a protein

target ligands and therefore the protein’s function

specificity and high affinity specificity: ability of a protein to preferentially bind to one or small number of molecules and not others high affinity: strength of binding between protein and ligand (strong interaction: protein and ligand associated for long time, weak interaction: protein and ligand come together and fall apart almost immediately) Both are dependent upon the molecular complementarity between the ligand and surface of the ligand binding site

molecular complementarity, which is dependent upon non-covalent interactions between facing surfaces

shapes are good matches and they fit close together and form many non-covalent interactions/shapes are poor matches and prevent them from getting close and forming non-covalent interactions

allows molecules to stay together despite the forces of thermal motion - based on complementarity of shape and molecular complementarity

when a protein interacts w/another molecule that is not another protein, we often refer to the ligand binding site as the ligand binding pocket has 6 specific aa resides with side chains facing the surface of pocket. aa’s cluster together and are essential in helping bind (increase affinity) cAMP within the pocket.