Molecular Biology & Biochemistry - Protein
These course outlines are a guide only. They are provided for the information of prospective students. Although every effort is made to ensure the most up to date information is provided, timetables often change each semester due to the availability of rooms and resources. Content (including lecture/practical topics, assessment and textbooks) is also regularly reviewed to ensure relevance and effective learning.
Most of the recent exciting progress in our understanding of the nature of living systems has come from a combined approach. Modern molecular biology techniques allow us to establish both the structure of genes (and hence the nature of their encoded proteins) and also how gene expression is regulated in response to different physiological stimuli. Biophysical techniques (notably NMR and X-ray crystallography) allow us to define the structure of macromolecules at the molecular level and so reveal important clues as to their functions.
These ideas will be illustrated in this course, which contains accounts of how proteins fold to their native state and are sorted to their desired destinations. You will be introduced to the basics of the important techniques in structural biology and learn how these, when combined with molecular biology, give us unparalleled scope for understanding the molecular basis of life.
Mrs Jill Johnston
Telephone: 9351 4248
Prof Joel Mackay
Telephone: 9351 3906
[MBLG (1001 or 1901) and 12 CP of Intermediate BCHM/MBLG units (taken from MBLG2071/2971 or BCHM2071/2971 or BCHM2072/2972)] OR [42CP of Intermediate BMedSc units (taken from BMED2801, BMED2802, BMED2803, BMED2804, BMED2805, BMED2806, BMED2807, BMED2808, but including BMED2802 and BMED2804)] OR [18CP of intermediate BMED units (taken from BMED2401, BMED2402, BMED2403, BMED2404,BMED2405, BMED2406, but including BMED2401 and BMED2405) and 6CP of Intermediate BCHM/MBLG units (taken from MBLG2071/2971 or BCHM2071/2971)
MBLG (1001 or 1901) and Distinction in 12 CP of Intermediate BCHM/MBLG units (taken from MBLG2071/2971 or BCHM2071/2971 or BCHM2072/2972)] OR [42CP of Intermediate BMedSc units (taken from BMED2801, BMED2802, BMED2803, BMED2804, BMED2805, BMED2806, BMED2807, BMED2808, with Distinction in BMED2802 and BMED2804)] OR [18CP of intermediate BMED units (taken from BMED2401, BMED2402, BMED2403, BMED2404, BMED2405, BMED2406, with Distinction in BMED2401 or BMED2405) and 6CP of Intermediate BCHM/MBLG units (taken from MBLG2071/2971 or BCHM2071/2971) with Distinction in MBLG2071/2971 or BCHM2071/2971
1st Lecture: Monday 9 am Eastern Avenue LT
2nd Lecture: Tuesday 9 am Eastern Avenue LT
Practical: Even weeks, 10:00am - 1:00pm Monday/Tuesday OR 10:00am - 1:00pm Wednesday/Thursday, according to Student Timetable (classes start in Week 2)*
VENUE: All practical classes will be in the Biochemistry 3 lab, Level 4, Biochemistry and Microbiology building, G08
*Note that it is possible to leave the practical class to attend a lecture in another subject, in which case the practical class will finish at 2:00pm.
For BCHM3081/3981, the recommended textbook is:
Williamson M How Proteins Work Mike Garland, 2012
Branden C & Tooze J Introduction to Protein Structure (2nd edition, Garland, 1999)
Liljas A Structural Aspects of Protein Synthesis (World Scientific Publishing, 2004)
Lodish H et al Molecular Cell Biology(6th edition, W H Freeman 2008)
Werth B The Billion Dollar Molecule (Touchstone, 1994)
Prof Jacqui Matthews
|Making and Breaking Proteins|
|SA||Dr Sandro Ataide||Protein targeting and Proteins in Cell Communication|
|JPM||Prof Joel Mackay||Protein Folding|
|Protein Folding and Disease|
|JPM||Prof Joel Mackay||
|JMM||Prof Jacqui Matthews|
|NS||Dr Nicholas Shepherd|
|CC||A/Prof Charles Collyer||Advanced course (Weeks 5 & 6, Tue 8am, Week 12 Tue & Thu 8am, Room 471)|
Making and Breaking Proteins
J Matthews: 3 lectures
1-2. Overview, Protein Synthesis Deciphering the genetic code. MRNAs, tRNA and Ribosomes. Activation of tRNAs. Ribosome assembly. Initiation, elongation and termination. Suppression of mutations. Protein secretion and folding. Regulation of protein synthesis. Inhibition of translation.
3. Protein Degradation Selective degradation of proteins. Cytosolic degradation: ubiquitination, proteosomes.
Protein targeting and Proteins in Cell Communication
S Ataide: 7 lectures
1. Secreted Proteins Overview of central dogma. Protein sorting pathways. Secretory protein in the ER. Signal recognition particle pathway. Translocon. Post-tranlsational translocation. Protein folding in the ER.
2. Membranes, membrane proteins and glycosylation Membranes and its properties. Membrane proteins. Insertion of membrane proteins. Membrane proteins topology. Glycosylation.
3. Disulfides and protein folding; sorting to specific organelles Formation of disulfide bonds. Protein folding in the ER. Sorting proteins to organelles. Import of mitochondrial proteins. Targeting sequences for mitochondria. Peroxisomes. Sorting to peroxisomes
4. Nuclear import and export Nuclear barrier. Nuclear pore complex. Nuclear localization sequence. Nuclear import. Nuclear export. Roles of importins. The Ran cycle.
5. Vesicles and vesicular trafficking Protein secretion. Vesicular traffic. Transport vesicles and the SNARE. Stages of secretory pathway. Structure of the Golgi. Clathrin and vesicle formation. Dynamin. Lysosomes. function, storage and trafficking.
6. Cytoskeleton Cytoskeleton. Filaments. Cell movement. Microfilaments and Actin. Fibroblast. Microtubules. Kinesin and Dynein. Intermediate filaments
7. Cell-cell & cell-matrix adhesion Cell adhesion molecules. Cell-cell adhesion. Collagen. Proteoglycans.
Protein folding; Protein Folding and Disease
J Mackay: 5 lectures
1. Fundamentals of protein folding Review of the fundamentals of protein folding. Anfinsen, Levinthal. Cooperativity of folding. Contributions to folding (electrostatic interactions, hydrophobic effect, conformational entropy). Denaturants. Mechanisms of folding (framework model vs hydrophobic collapse). Methods for examining folding. Partially folded intermediates. The folding funnel. Techniques: Near and far-UV CD, fluorescence, NMR.
2. Serpins Discussion of the mechanism through which the serpin family of proteins acts to inhibit serine proteases, as an example of the balance of kinetic versus thermodynamic control of protein folding. Serpinopathies.
3-4. Protein folding in the cell GroEL/GroES, disulphide bond isomerases, peptidyl-prolyl isomerases.
5. Diseases of protein folding Cystic fibrosis – demonstration that it is a problem with folding kinetics. Amyloidoses (transthyretin diseases, Alzheimers, prion diseases).
Protein Engineering and Design
J Mackay, N Shepherd and J Matthews: 8 lectures
1-2. Basics of protein design (JPM) Early T4 lysozyme experiments – introduction of SS bonds, salt bridges, repacking hydrophobic core to engineer thermostability. Secondary structure propensities, considerations in protein engineering and design. Peter Kim’s chameleon sequence. Rop and the Peracelsus challenge, Stephen Mayo’s inverse design. Computational prediction of protein structure, CASP. Techniques: site directed mutagenesis, computational protein design, making recombinant proteins (subcloning, affinity tags).
3-4. Making designer transcription factors (JMM) DNA binding proteins. How proteins recognize DNA. Random libraries and phage display, making designer zinc finger proteins, parallel vs series selection, examples. Zinc finger nucleases. Using ZNF libraries to select for a phenotype (with no knowledge of gene you are targeting). Techniques: Making random libraries, phage display
5-6. Protein design, modification and mimicry (NS) Protein Design: Rational directed evolution. In-vitro protein evolution. Protein modification: incorporating unnatural amino acids into proteins using recombinant and semi-synthetic methods. Protein bioconjugation methods and applications. Protein mimicry - replicating protein surfaces using mini-proteins and peptide fragments. Techniques: mRNA display, Amber stop-codon suppression, intein mediated protein splicing, native chemical ligation.
7-8. Protein interactions on the organism wide scale (JMM) Detecting and analyzing protein interaction networks within an organism. Comparison between organisms. Techniques: Tandem affinity purification, mass spectrometry, yeast two-hybrid, including large-scale yeast two-hybrid screens.
P1 Protein Bioinformatics (1 week)
P2 Overexpression of a cloned gene (3 weeks)
P3 Purification of Replication Termination Protein from B.subtliis (2 weeks)
P4 DNA-Protein interaction and the Band Retardation Assay (1 week)
Lecture course: 50% (end-of-semester examination)
Practical course: 50% (25% in-semester practical work, 25% end-of-semester examination)