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Lecture 6

BIOC12 Lecture 6.doc

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University of Toronto Scarborough
Biological Sciences
Shelley A.Brunt

BIOC12 Lecture 6 Slide 2 Quaternary Structure -identical polypeptide chains are called protomers, also known as (aka) multiple monomers -subunits are held together by weak non-covalent bonds, primarily hydrophobic -tight association provides stable structure, but the interaction cannot be too strong to allow separation of subunits -quaternary (4 ̊ structure is more stable than individual subunit or tertiary (3̊ structure, regardless if the individual subunit has same or different polypeptide chains as the 4̊ structure -4̊ structure has a greater half life because it is more stable -interior (inside) of a single subunit is hydrophobic, folds to the center (core) -contact regions (interactive area between different subunits) are also hydrophobic -in some cases, one subunit does not contain the substrate binding site -different proteins can share same subunits, meaning they have identical subunits that allow the same ligand to bind -ex. if J binds to A & B subunits and also C & D subunits, the identity of J is a ligand -since ligand J can be shared between the two sets of subunits, it means subunits A & B and C & D are identical Slide 3 Chicken Triose Phosphate Isomerase -quaternary structure with 2 identical subunits Slide 4 Tubulin -globular protein -heterodimer (aka microtubule subunit), consists of α-tubulin and β-tubulin -protofilament is a linear row of heterodimer, also an example of multimeric (oligomeric) protein -function: 1) play a role in making microtubules & cytoskeleton structure -microtubule is the overall cylindrical shape -2) move vesicle, granules, organelles (mitochondria & chromosome) via special attachment protein pulling of chromosome in mitosis & meiosis -tubulin is added to the + end & lost in the – end Slide 5 haemoglobin -tetrameric, four substrate binding sites -myoglobin: 1 subunit of haemoglobin not a good oxygen carrier -lack of oxygen, muscle gets tired more easily than blood Slide 6 Immunoglobulin (IgG) -Y shaped, consists of two identical heavy chains & two identical light chains (tetrameric quaternary structure) -each end of the fork is the antigen binding site, 2 antigens -the other end (FC region) attaches to phagocytic cell for disposal -constant region interacts with Fragment Crystallizable (FC) region -many disulfide bonds within light and heavy chains, linking them as well -secretory place is the extracellular matrix and circulating blood, which is highly oxidized disulfide bonds can stabilize the structure in the extracellular matrix -for secretory protein: addition of carbohydrate (glycosylation) contributes to its stability in extracellular matrix -cytosol is a reduced environment, harder to form disulfide bonds Slide 7 IgG -more beta sheet than alpha helix to stabilize the structure Slide 8 Table of Natural Occurrence of Oligomeric Proteins in Escherichia coli -most proteins are dimers, around 40% -monomer & tetramer are both around 20% each Slide 9 Macromolecular Assemblies -macromolecular assemblies: make up of many compartments (ribosome, spliceosome, nuclear pore complex, transcription initiation complex, chaperonins), not just proteins -ribosome is multimeric protein does not function on its own, higher oligomeric state -in bacteria, flagella consists of many proteins, higher oligomeric state as well -chaperonin is individual protein found in cytosol & mitochondria -proteosome: degrade proteins Slide 10 Large Protein Complexes in Mycoplasma pneumonia -Mycoplasma pneumonia is the smallest genus -can survive without oxygen -still require ribosome & chaperonin GroEL to function -most proteins are dimer, including transcription factor -do not work on their own, need help from other proteins with folding & traffic Slide 11 Bacterial Flagellum -motor at the bottom -consists of 21 different proteins Slide 12 E. coli Interactome -protein sequence is analyzed to generate interactome, but post-translational mechanism makes it harder -shows the interactions of the proteins, not all proteins interact at the same time Slide 13 Protein Stability and Folding -hydrophobic effect, hydrogen bond, and metal ions contribute to stability of protein, but hydrophobic effect plays the greatest role -electrostatic interactions are important in region that is not exposed to water, otherwise it interacts with water instead Slide 14 Hydropathy Plot of Bovine Chymotrypsinogen -interaction with metal ion: zinc finger helps stabilize structure for DNA binding with transcription factor Slide 15 Amino Acid Sequence of a Protein Determines Three Dimensional Structure -function of ribonuclease A is to hydrolyze RNA -hard to isolate RNA in lab experiment -keep in mind that denaturation does not equal to degradation -denaturation: unfolding, break down to amino acid sequence (primary structure) renaturation resumes when the chemical/ environmental change is removed -degradation: falling apart, break the proteins when subunits fall apart Slide 16 Thermal Denaturation Curve of RNase A -Tmis the temperature at which half of the proteins unfold/ denature -interior of a protein stabilizes its structure, destroy those hydrogen bonds first to unfold the protein -need to break 3-4 hydrogen bonds first in order to unfold the protein Slide 18 Chaotropic Agents -chaotropic agents: denature proteins -ex. urea, guanidinium chloride, detergents -do not use sodium dodecyl sulfate (SDS) because it is hard to remove them, which makes renaturation impossible Slide 25 Denaturation/ Renaturation Cycle of RNase A -addition of urea & mercaptoethanol denatures the protein -protein renatures when urea & mercaptoethanol are removed -however, if mercaptoethanol is removed, but urea remains, the protein forms disulfide bonds randomly that is not in the right order, native state of protein is not returned in order for the protein at this stage to return to its native state, urea is removed, mercaptoethanol is added along with some heat -therefore, both urea & mercaptoethanol must be removed in order for the protein to renature Slide 27 Protein Disulfide Isomerase (PDI) -two types of PDI 1) Reduced PDI: rearranges non-native disulfide bonds via reduction of disulfide bonds, help correct protein folding 2) oxidized PDI: helps in forming disulfide bonds Slide 30 -in protein folding, high energy is required at the beginning -energy well is wider and narrower in final stage (slide 31) -protein needs to be folded very quickly at the beginning to prevent degradation Slide 31 -bumps in the energy well indicate that protein folding is a back & forth folding, folding & unfolding -non-linear, not progressive step-by-step folding Slide 33 Three Stages in Unassisted Protein Folding -2 stage: folding of molten globule (intermediate), secondary structures are present -small protein can fold on its own without chaperone, weak individual bonds, easy to break Slide 37 Models for Protein 1) Framework Model -α-helix & β-pleated sheet (2̊ structure) fo
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