MIT 5.08J: Biological Chemistry II
Instructor: Professor Elizabeth Nolan, MIT Department of Chemistry
This comprehensive lecture series covers the fundamental mechanisms of biological chemistry, from protein synthesis and folding to degradation pathways and complex biosynthetic machinery. The course provides deep insights into how cells synthesize, fold, and degrade proteins, as well as the intricate assembly lines that produce polyketides and nonribosomal peptides.
Module 1: Translation
The molecular machinery of protein synthesis, including ribosome structure, tRNA charging, translation initiation, elongation, and termination.
1. Introduction to Biological Chemistry II
Course overview, introduction to the central dogma, and preview of protein synthesis, folding, degradation, and biosynthetic pathways.
2. Protein Synthesis 1
Introduction to the ribosome, tRNA structure and aminoacylation, the genetic code, and the wobble hypothesis.
R1. Determining, Analyzing, and Understanding Protein Structures
Methods for determining protein structures: X-ray crystallography, NMR, cryo-EM. How to analyze and interpret structural data from the Protein Data Bank (PDB).
3. Protein Synthesis 2
Translation initiation in prokaryotes: initiation factors (IF1, IF2, IF3), ribosome assembly, and formation of the 70S initiation complex.
4. Protein Synthesis 3
Translation elongation: EF-Tu and EF-G cycles, peptidyl transferase mechanism, ribosome translocation, and proofreading mechanisms ensuring fidelity.
5. Protein Synthesis 4
Translation termination, release factors (RF1, RF2, RF3), ribosome recycling, and polysome dynamics.
R2. Pre-Steady State and Steady-State Kinetic Methods Applied to Translation
Rapid kinetic techniques for studying translation: quench-flow, stopped-flow, fluorescence methods. Distinguishing pre-steady state from steady-state kinetics.
6. Protein Synthesis 5
Eukaryotic translation initiation, the cap-binding complex, scanning mechanism, and regulation by phosphorylation of initiation factors.
7. Protein Synthesis 6
Antibiotics targeting the ribosome, resistance mechanisms, and the use of ribosome inhibitors as tools to study translation.
Module 2: Protein Folding
Thermodynamics and kinetics of protein folding, Anfinsen's principle, Levinthal's paradox, energy landscapes, molecular chaperones, and protein misfolding diseases.
8. Protein Folding 1
Introduction to protein folding: primary through quaternary structure, forces stabilizing folded proteins, and the thermodynamics of folding. Anfinsen's principle and Levinthal's paradox.
R3. Pre-Steady State and Steady-State Kinetic Methods Applied to Translation
Application of kinetic methods to study biological processes. Essential background for understanding protein folding kinetics and experimental approaches.
9. Protein Folding 2
Energy landscapes, folding funnels, and kinetic pathways. Discussion of molten globule states, folding intermediates, and the role of conformational entropy in guiding folding.
10. Protein Folding 3
Experimental methods for studying protein folding: circular dichroism, fluorescence spectroscopy, hydrogen-deuterium exchange, and NMR. Introduction to chaperones and protein quality control.
11. Protein Folding 4
Molecular chaperones (GroEL/GroES, Hsp70, Hsp90), protein misfolding diseases (Alzheimer's, Parkinson's, prion diseases), and the cellular protein quality control system. Proteostasis and aggregation.
Module 3: Protein Degradation
The ubiquitin-proteasome system, protein quality control, targeted protein degradation, and the role of post-translational modifications in regulating protein stability.
R4. Purification of Native and Mutant Ribosomes, Protein Purification
Methods for purifying ribosomes and proteins: affinity chromatography, ion exchange, size exclusion, and techniques for isolating mutant ribosomes for functional studies.
12. Protein Degradation 1
Introduction to the ubiquitin-proteasome system, ubiquitin structure, E1-E2-E3 enzyme cascade, and polyubiquitin chain formation.
13. Protein Degradation 2
The 26S proteasome structure and mechanism, substrate recognition, deubiquitination, unfolding, and proteolysis in the catalytic core.
14. Protein Degradation 3
Regulation of protein degradation, degrons, N-end rule, PROTACs (proteolysis-targeting chimeras), and therapeutic applications of targeted protein degradation.
R5. Overview of Cross-Linking, Including Photo-Reactive Cross-Linking Methods
Chemical and photo-reactive cross-linking methods for studying protein-protein interactions, protein complexes, and conformational dynamics. Mass spectrometry analysis of cross-linked peptides.
Module 4: Polyketide and Nonribosomal Peptide Assembly Lines
Biosynthesis of complex natural products by modular enzyme systems. Polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) create diverse bioactive compounds including antibiotics.
15. PK and NRP Synthases 1
Introduction to polyketide synthases: fatty acid synthase (FAS) as a model system, domain organization, acyl carrier protein (ACP), and the chemistry of chain elongation.
16. PK and NRP Synthases 2
Modular PKS systems: erythromycin synthase, module structure, ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains controlling stereochemistry.
R6. Macromolecular Electron Microscopy Applied to Fatty Acid Synthase
Cryo-electron microscopy (cryo-EM) for determining structures of large macromolecular assemblies like fatty acid synthase. Single-particle analysis and image reconstruction.
R7. Application of Single Molecule Methods
Single-molecule fluorescence (smFRET), optical tweezers, and atomic force microscopy (AFM) for studying enzyme dynamics, conformational changes, and mechanical properties of proteins.
17. PK and NRP Synthases 3
Nonribosomal peptide synthetases (NRPS): adenylation (A), thiolation (T), and condensation (C) domains. Examples: gramicidin S, cyclosporin, and vancomycin biosynthesis.
18. PK and NRP Synthases 4
Hybrid PKS-NRPS systems, combinatorial biosynthesis, rational design and engineering of assembly lines for novel natural product synthesis. Applications in drug discovery.
Module 5: Cholesterol Biosynthesis and Cholesterol Homeostasis
The intricate pathways of cholesterol biosynthesis, regulation, and cellular homeostasis mechanisms. From acetyl-CoA to cholesterol structure, and the sophisticated regulatory networks that control cholesterol levels.
R7. Application of Single Molecule Methods
Single-molecule techniques for studying biomolecular processes: FRET, optical tweezers, and total internal reflection fluorescence microscopy (TIRF). Applications to protein folding and enzymatic mechanisms.
19. Cholesterol Biosynthesis 1
Overview of cholesterol biosynthesis pathway. From acetyl-CoA to HMG-CoA to mevalonate. The committed step catalyzed by HMG-CoA reductase and its regulation.
20. Cholesterol Biosynthesis 2
From mevalonate to isopentenyl pyrophosphate (IPP). Condensation of IPP units to form squalene. Cyclization of squalene to lanosterol by oxidosqualene cyclase.
21. Cholesterol Biosynthesis 3 & Cholesterol Homeostasis 1
Conversion of lanosterol to cholesterol (19 enzymatic steps). Introduction to cholesterol homeostasis: SREBP pathway, SCAP, and Insig proteins. Regulation by cellular cholesterol levels.
R8. Application of CRISPR to Study Cholesterol Regulation
CRISPR-Cas9 genome editing for functional genomics studies. Applications to dissecting cholesterol regulatory pathways. Knockout screens and genetic perturbation approaches.
22. Cholesterol Homeostasis 2
LDL receptor pathway: receptor-mediated endocytosis of cholesterol-rich lipoproteins. Clathrin-coated pits, endosomes, and lysosomal cholesterol release.
23. Cholesterol Homeostasis 3
NPC1/NPC2 proteins and lysosomal cholesterol export. Niemann-Pick disease. Cholesterol trafficking to the ER and other cellular membranes.
24. Cholesterol Homeostasis 4
SREBP processing and activation. Site-1 and Site-2 proteases (S1P, S2P). Nuclear translocation and transcriptional activation of cholesterol biosynthesis genes.
R9. Cholesterol Homeostasis and Sensing
Mechanisms of cholesterol sensing by SCAP and Insig proteins. Conformational changes in response to ER cholesterol levels. Integration of multiple regulatory inputs.
25. Cholesterol Homeostasis 5 & Metal Ion Homeostasis 1
Summary of cholesterol homeostasis mechanisms. Transition to metal ion homeostasis: essential metals in biology (Fe, Cu, Zn, Mn), requirements and toxicity.
Module 6: Metal Ion Homeostasis
The sophisticated systems cells use to acquire, transport, store, and regulate essential metal ions. Mechanisms preventing metal toxicity while ensuring sufficient availability for metalloenzymes and cofactors.
26. Metal Ion Homeostasis 2
Iron homeostasis: transferrin, transferrin receptor, and cellular iron uptake. Ferritin and iron storage. Iron-responsive elements (IREs) and iron regulatory proteins (IRPs).
27. Metal Ion Homeostasis 3
Post-transcriptional regulation by IRE/IRP system. Hepcidin and systemic iron regulation. Hemochromatosis and iron overload diseases.
R10. Metal-Binding Studies and Dissociation Constant Determination
Experimental methods for studying metal-protein interactions. Isothermal titration calorimetry (ITC), fluorescence quenching, competition assays. Determining binding stoichiometry and affinity constants.
28. Metal Ion Homeostasis 4
Copper homeostasis: copper transporters (CTR1), copper chaperones (ATOX1, CCS, COX17). Menkes and Wilson diseases (ATP7A/ATP7B mutations).
29. Metal Ion Homeostasis 5
Zinc homeostasis: ZIP and ZnT transporter families. Zinc finger proteins and structural zinc sites. Metallothioneins and metal detoxification.
R11. Mass Spectrometry
Mass spectrometry fundamentals: ionization methods (ESI, MALDI), mass analyzers (TOF, quadrupole, orbitrap). Protein identification, post-translational modifications, and quantitative proteomics.
30. Metal Ion Homeostasis 6
Manganese homeostasis and transporters. Transcriptional regulation of metal homeostasis genes. Metal-sensing transcription factors and riboswitches.
31. Metal Ion Homeostasis 7 & Reactive Oxygen Species 1
Summary of metal homeostasis principles. Introduction to reactive oxygen species (ROS): superoxide, hydrogen peroxide, hydroxyl radical. Sources of cellular ROS.
Module 7: Reactive Oxygen Species (ROS)
The double-edged sword of reactive oxygen species: their generation, cellular damage, defense mechanisms, and roles in signaling. From oxidative stress to redox regulation of protein function.
R12. Mass Spectrometry of the Cysteine Proteome
Redox proteomics: identifying oxidatively modified cysteine residues. Isobaric tagging, enrichment strategies, and quantitative analysis of cysteine oxidation states across the proteome.
32. Reactive Oxygen Species 2
Cellular antioxidant defense systems: superoxide dismutase (SOD), catalase, glutathione peroxidase. Glutathione (GSH) and thioredoxin systems. Enzymatic mechanisms and metal cofactors.
33. Reactive Oxygen Species 3
Oxidative damage to biomolecules: lipid peroxidation, DNA oxidation (8-oxo-guanine), protein carbonylation. Cellular consequences of oxidative stress and repair mechanisms.
34. Reactive Oxygen Species 4 & Nucleotide Metabolism 1
Redox signaling and regulation: Nrf2/Keap1 pathway, peroxiredoxins, and redox-sensitive transcription. Transition to nucleotide metabolism: overview of purine and pyrimidine biosynthesis.
Module 8: Nucleotide Metabolism
The biosynthesis, salvage, and degradation pathways of purine and pyrimidine nucleotides. Regulation of nucleotide pools, clinical relevance, and therapeutic targeting of nucleotide metabolism.
R13. Fluorescence Methods
Fluorescence spectroscopy principles: excitation, emission, quantum yield, and fluorescence lifetime. FRET, anisotropy, and applications to studying biomolecular interactions and conformational changes.
35. Nucleotide Metabolism 2
De novo purine biosynthesis: the 10-step pathway from PRPP to IMP. Committed step catalyzed by glutamine PRPP amidotransferase. Conversion of IMP to AMP and GMP. Regulation by feedback inhibition.
36. Nucleotide Metabolism 3
Pyrimidine biosynthesis: CAD multi-enzyme complex, dihydroorotate dehydrogenase, UMP synthase. Salvage pathways (HGPRT, APRT). Clinical aspects: gout, Lesch-Nyhan syndrome, and anticancer/antiviral drugs targeting nucleotide metabolism.
Course Impact
Professor Elizabeth Nolan's MIT 5.08J course provides a comprehensive foundation in biological chemistry, bridging molecular mechanisms with physiological function. The course emphasizes both the chemical principles underlying biological processes and the experimental techniques used to elucidate them.
Key Topics Covered (36 Lectures)
- Ribosome structure and translation mechanism
- Protein folding thermodynamics and kinetics
- Molecular chaperones and quality control
- Ubiquitin-proteasome degradation system
- Polyketide and nonribosomal peptide biosynthesis
- Cholesterol biosynthesis and homeostasis (SREBP pathway)
- Metal ion homeostasis (Fe, Cu, Zn, Mn)
- Reactive oxygen species and antioxidant defense
- Purine and pyrimidine nucleotide metabolism
Experimental Techniques (13 Recitations)
- X-ray crystallography and cryo-EM structure determination
- Rapid kinetic methods and pre-steady-state kinetics
- Single-molecule fluorescence (FRET, TIRF)
- Cross-linking mass spectrometry and proteomics
- CRISPR-Cas9 genome editing for functional genomics
- Isothermal titration calorimetry (ITC) for binding studies
- Mass spectrometry (ESI, MALDI, orbitrap)
- Redox proteomics and cysteine oxidation profiling
- Fluorescence spectroscopy and anisotropy