bibc 100 handout 4
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BIBC 100 – Structural Biochemistry TA: Lydiesther MartinezDr. Montal Lymartin@ucsd.eduSpring ’11 Section: Mon 7pm
WEEK 4 and 5Protein Folding Introduction
o Problem: protein folding is statistically impossible if it is completely ____________o Levinthal’s paradox – proteins usually fold in 10-1 to 103 seconds
o Conclude that folding is not ____________, but deterministico USA – ____________ (action), ____________ (energy), ____________ (kinetics)
o Important because folding is 2nd genetic code – could lead to computer designed proteins
Anfinsen Experiment Tested ribonuclease to see if protein folding was dictated by the ____________sequence Reagents:
o ____________– interact with water and other molecules to disrupt hydrophobic interactionso -____________– reducing agent to break disulfide bonds
Found that primary sequence was enough for small globular proteins to fold into ____________state
Free Energy Funnel for Protein Foldingo Entropy – drives towards unfolded state with hydrophobic residues
exposedo ____________– drives toward folded state with hydrophobic
interactions, H bonding, ionic interactionso Free energy is the difference – drives towards folded stateo Common obstacles to folding:
____________of hydrophobic regions Non-native ____________bridge formation ____________of proline
o Unfolded molten globule (loose 3° structure) is fasto Molten globule native structure is slowero Some proteins never reach their native stateo Other stable structures are not functional
transition state between molten globule and native state requires ____________in free energy
Prions & Alzheimer’s Diseaseo Prion – “proteinaceous infectious only” o PrPc (prion protein) is a normal constituent of cells with significant helix contento Changes in conformation allow____________ content (PrPsc) propagates to
other proteins forms amyloid fibers. Fibers accumulate in nerve cells, which spend all their energy trying to get rid of the toxin lose homeostatic energy
o Results in loss of memories death
Protein Folding Chaperones – GroEL/GroES Complex ____________ (HSP) – prevent damage to proteins in response to high levels of
heat by preventing aggregation of exposed hydrophobic regionso Nonspecific – recognizes exposed hydrophobic patches
HSP 70: regulates translocation to the ER, assembly & disassembly of oligomers GroEL (Hsp 60): 7 subunits with apical (______), intermediate (___), equatorial domains (____) GroES (Hsp 10): ___ hairpin roof – sheet – mobile _______(integrated with GroEL) GroEL/ES complex has 2 barrels end-to-end, so each can accommodate a protein but GroES cap is an
BIBC 100 – Structural Biochemistry TA: Lydiesther MartinezDr. Montal Lymartin@ucsd.eduSpring ’11 Section: Mon 7pm
__________ inhibitor for the other barrel
Protein Stability o Can detect through _________ (site-directed protein engineering)o Disulfide (S-S) bridges: reducing the entropy through covalent bonds increases protein stabilityo G & P: Glycine is much more flexible than Proline (side chain is covalently bonded to main chain)
o G P or A increases stabilityo Dipole stability: N terminus should have (-); C terminus should have (+) to increase stabilityo Hydrophobicity in core cavity increases stability
(thermodynamics)
Allosteric Control o Allosteric effectors: small ligands that bind to
_________ _________ recognition sites (away from functional binding sites)
o Trp repressor controls operon that synthesizes L-trpo Absence of trp: helices tilt inward - 29Å distance
between binding sites (inactive repressor)o trp bound (2) recognition helices ______ apart for proper binding of DNA (active repressor)
CAP – catabolite gene activating protein Motif: _________ _________ DNA binding protein that assists RNA polymerase in effective binding
o Promotes initiation of RNA synthesis Functions as _________ Activated by _________ (allosteric effector) Interacts directly with base pairs & phosphate groups Bending of DNA contributes to activation of transcription _____ – mutations have been associated with many diff types of cancer
(WT is tumor suppressor by controlling cell cycle)o DNA binding protein interaction of helix with groove of DNAo H-bonds between protein & base pairs
Zinc Finger Motif o Motif: 1 _________ & 2 _________ coordinated by a ____ metal
(stabilized by 2 Cys, 2 His – midterm 6a) Approximately 30 amino acids Highly conserved coordination sites (midterm 6b)
BIBC 100 – Structural Biochemistry TA: Lydiesther MartinezDr. Montal Lymartin@ucsd.eduSpring ’11 Section: Mon 7pm
Proteins contain anywhere from 1-60 zinc finger motifs within sequence alpha helix side chains _________ bond with base pairs of DNA (midterm 6c) Fig 10.3 – schematic diagram of 3 Zn fingers binding in tandem to major groove – surround DNA
Leucine Zipper Motif Motif: _________ _________ alpha helices Heptad repeat: almost all 4th residue is _________ (fig 10.17)
o Character of AA repeat every ___ AA _________ core is major contributing factor to stability Distal, basic region on each helix interacts with major groove
o Increased concentration of _________o Likely AA: _________ and _________
Immune Response Immune system: recognizes, destroys, and remembers _________ _________
while ignoring _________ cells & proteins Antigen recognizes foreign molecules immune response
o ____ cells release immunoglobulins to circulationo ____ cells have cell receptor that recognizes _________ protein
Immunoglobulin Fold Heterotetramer: 2 ____ chains + 2 _____ chains
o Linked by ____ disulfide bonds stabilityo Hinge region of HC _________ (Y/T conformations)
Ig fold is distorted _________o Each LC contains _____ Ig folds, each HC contains ___ Ig foldso Distorted conformation stabilized by _____ disulfide bridges
Antigen recognition at loops between strands at N-terminus of Ig Each Ig recognizes ___ antigen(s)
o Different B cells diff Igo Heavy & light chains are encoded by different geneso N terminus regions contain the most variabilityo Hypervariable loops provide antigen recognition & specificity
Bind to antigen via _________ bonds Papain cleaves _________ bonds and separates the IgG into 2 F__ sites (light & heavy chain – antigen
binding site) and F___ (2 heavy chains)
Good luck!
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