Protein and Nucleic Acid Chemistry
Winter 2003

Biotechnology 3P63 Prof. J. Atkinson


Page numbers in bold are from Voet and Voet's Biochemistry, 2cd Edition

Basic Nucleic Acid Structure (Chapter 28)

 o bases, phosphate esters, ribose sugars
 o helix dimensions and conformations
 o H-bonds; Watson-Crick, Hoogsteen
 o stuctural types (B, Z, A, supercoiled, hairpin loops, etc)
 o DNA structure/function
 o hybridization
Chemical DNA Synthesis and Sequencing
o solid phase phosphoramidate methodology
o sequencing: chemical means, dideoxynucleotides (Sanger method)
o chemcial reactions of DNA with small molecules

 

 

DNA Damage and Repair (p. 1046-1051)
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Introductory Chemistry of Proteins (Chapters 4 and 5)

o amino acids, stereochemistry, pKas
Primary sequence information (Chapter 6)
o primary sequences, genetic code(s), information handling
o overview of biosynthesis: transcription and translation
o protein transport and targeting (p. 312-314)
o post-translational modifications
- glycosidation
- N-terminal amidation (glycine oxidase), pyroglutamylation
- lipid conjugation: myristoylation, farnesylation and membrane insertion
o ubiquination and protein recycling / degradation (p. 1010-1014)
Secondary and Tertiary Structure (Chapters 7 and 8)
o H-bonding, strength and variation, hydrophobic interactions
o secondary motifs - 1° stucture and 2° structure prediction (hydropathy, amphipathicity, etc)
- salt-bridges, disulphide bonds (R-S-S-R)
- helices, sheets, turns, random coil, etc.
o tertiary motifs - Greek keys, b-barrel, TIM barrels, etc
o protein folding (Chapter 8)
o structure and function i.e. membrane spanning proteins, ion channels, collagen, certain enzymes,
Protein Modification
o labeling and affinity labeling
- haloacetaes, RCHO/NaBH4, iodination, oxidation
- photoaffinity labeling
- ELISA's and antibody technology

 

 

Structure Determination

o How to read and interpret x-ray stuctures
- resolution, R-factors, rms deviations
- NMR and MS of proteins
Labs
1. Comparison of chemical and spectroscopic means of protein and DNA detection. Absorbance ratios for mixtures of proteins and DNA. Colourometric tests using dye binding (Biorad) and Pierce assay systems. Standard curve construction for proteins of varying chemistries.
2. Isolation of supercoiled plasmid DNA, and the analysis of the extent of coil relaxation by visualization of multiple bands after electrophoresis in chloroquine containing gels.
3. Assessment of DNA methylation status by methylation-sensitive restriction endonucleases
4. The effect of denaturants on the measured activity of the thiol-proteinase papain
5. Lectin / glycopeptide anaylsis using Boehringer-Manheim kit. Illustrates post-translational modification by sugars and their detection by the use of lectins, and specific enzyme linked antibodies in a colourometric test.
6. Protein electrophoresis, Western blot and immunodetection of various serum albumins.

Instructions for the timing of handing in laboratory reports and late penalties will be arranged with your demonstrator. A student must attend a minimum of four of the six labs in order to pass the course.

Term Project Use of protein visualization and anlaysis software to inspect primary sequence data and assess secondary structure predictions of a published x-ray crystal structure. More details will be provided in class.

Marking Scheme

Assignments (3) 15%
Labs (6)  25%
Term Project 10%
Midterm 15%
Final 35%

Late assignments will be penalized at a rate of 30% per day late. After three late days, assignments and projects will not be accepted.