BCHM3081/3981

Molecular Biology & Biochemistry - Protein

Course Information

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

Room: 410

Telephone: 9351 4248

FAX: 9351 4726

E-mail: j.johnston@usyd.edu.au

A/Prof Joel Mackay

Room: 605

Telephone: 9351 3906

FAX: 9351 4726

E-mail: j.mackay@usyd.edu.au

For BCHM3081
(MBLG (1001 or 1901) and 12 CP of Intermediate BCHM/MBLG units (taken from MBLG2071/MBLG2971or BCHM2071/2971 or BCHM2072/2972)) or (42CP of Intermediate BMedSc units, including BMED2802 and BMED2804)

For BCHM3981
MBLG (1001 or 1901) and Distinction in 12 CP of Intermediate BCHM/MBLG units (taken from MBLG2071/MBLG2971 or BCHM2071/2971 or BCHM2072/2972) or 42CP of Intermediate BMedSc units, with Distinction in BMED2802 and BMED2804.

1st Lecture: Wednesday 9 am Carslaw Lecture Theatre 275

2nd Lecture: Friday 9 am Merewether Lecture Theatre 2

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:

Branden C & Tooze J Introduction to Protein Structure (2nd edition, Garland, 1999)

OR

Nelson, D L & Cox, M M Lehninger Principles of Biochemistry (5th edition, Freeman, 2008)

Reference texts

Liljas A Structural Aspects of Protein Synthesis (World Scientific Publishing, 2004)

Werth B The Billion Dollar Molecule (Touchstone, 1994)

Making and Breaking Proteins
J Matthews: 4 lectures

1-3. 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. Non-natural amino acids.

4. Protein Degradation. Selective degradation of proteins. Cytosolic degradation: ubiquitination, proteosomes

Introduction to Protein Folding
J Mackay: 2 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.

Protein Folding and Disease
J Mackay and M Sunde: 4 lectures

1-2. Protein Folding in the Cell. GroEL/GroES, disulphide bond isomerases, peptidyl-prolyl isomerases.

3-4. Diseases of Protein Folding. Cystic fibrosis – demonstration that it is a problem with folding kinetics. Amyloidoses (transthyretin diseases, Alzheimers, prion diseases). Techniques: Light scattering, fibre diffraction

Protein Engineering Design
J Mackay and J Matthews: 6 lectures

1-2. Basics of Protein Design. 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. 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. Developing Proteins Agonists/Antagonists. Growth hormone: recombinant growth hormone, interaction of GH with its receptors. Design of EPO antagonists/agonists. Techniques: Recombinant protein production/expression ITC, Biacore, gel filtration.

6. Protein Interactions on the Organism Wide Scale. 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

Protein Targaret
P Kuchel: 8 lectures

Membranes.
membrane structure and transport across membranes, SNARE proteins, clathrin-coated pits
Protein targeting.
import and export of proteins from the nucleus
roles of importins, the nuclear pore complex and the Ran cycle
delivery of proteins to cellular compartments (translocons, protein folding in the ER, including ER chaperones)
processing of proteins in cellular compartments (folding and glycosylation, processing in lysosomes)
vesicle trafficking, delivery of proteins to the outside of the cell, delivery of proteins to cellular compartments (mitachondria and peroxisomes)
ion channels, GLUT4 transporters, phagocytosis and receptor-mediated endocytosis, illustrated with LDL receptor
toxins

P1 Protein Bioinformatics
P2 Overexpression of a cloned gene
P3 Purification of the Overexpressed Protein
P4 Functional Studies on the Overexpressed Protein

Lecture course: 50% (end-of-semester examination)
Practical course: 50% (25% in-semester practical work, 25% end-of-semester examination)