BCH 3052: Protein Biology: from Sequence to Structure and Disease

• Convenor: Prof Mibel Aguilar 
• 6 points
• Monash Handbook

Lecturing Staff

Organisation of the unit

BCH3052 consists of 2 lectures per week and one 3 hour practical session per week and 1 tutorial every week. During the tutorials various assignments will be set which will aid student's understanding of the course.

Topics covered

CONFORMATIONAL CHANGE IN PROTEINS AS AN ESSENTIAL PART OF FUNCTION 

Tertiary structure of proteins and examples of conformational change as an important mechanism in normal function
Revision of amino acid groupings.  The 4 levels of protein structure.  Tertiary structure in proteins. Change in tertiary structure as an effector of function.  Examples of proteins where conformational change is important in function: calmodulin.

Conformational change in the mechanism of action of serpins
The structure of serine protease inhibitors (serpins).  The mechanism of action of serpins and the importance of conformational change.  Antithrombin as an important serpin involved in controlling blood coagulation.  The role of heparin in modulating the action of antithrombin.  Mutations in antithrombin structure cause deep vein thrombosis.

STRUCTURE AND ACTIVITY OF ENZYMES AND PROTEINS 

Enzyme kinetics: importance in drug design
Revision of basic enzyme kinetics.  What do the constants derived from enzyme kinetics mean in terms of physiological function?  The basic parameters underlying inhibition of enzymes.  The meaning of inhibition constants: what do they tell us?  The importance of inhibition constants in the design of enzyme inhibitors to combat disease.

Allosteric enzymes: importance in health and disease
Allosteric enzymes: models of allosteric behaviour.  Regulation of allosteric enzymes.  Positive and negative co-operativity.  Kinetic characterisation of allosteric behaviour and models.  Examples of allosteric behaviour in relation to physiological function and pathophysiological aberration.

STRUCTURAL CHARACTERISATION OF PROTEINS

Introduction to the structural features of macromolecules.
Macromolecules are composed of a number of different secondary, tertiary and possibly quaternary structures that dictate their function. Two key examples are explored. ATP synthase is the enzyme that synthesises ATP from ADP and inorganic phosphate. The enzyme has many features that are analogues to a machine or motor. The synthesis of ATP is central to life as it is utilized as a general currency of energy within all cells. Our muscles allow us to move, breathe and live our lives. How do our muscles utilize ATP? 

Structure determination using X-ray crystallography.
How are macromolecular structures determined? What is the primary technique utilized to determine the shape of a macromolecule to atomic resolution? The concepts underlying X-ray crystallography will be introduced. A practical guide to X-ray crystallography will be given. The key challenges in determining a crystal structure will be highlighted. The key statistics that describe the quality of a crystal structural as reported in the literature will discussed.

PROTEIN BIOENGINEERING & BIOTECHNOLOGY

Structure determination using NMR spectroscopy
A second method, after X-ray crystallography, for the determination of the three-dimensional structure of macromolecules is NMR spectroscopy. This lecture will explain the principles behind NMR spectroscopy and how it is used in macromolecular structure determination and for the detection of protein interactions with binding partners.

Peptide discovery and development as drug leads
Peptides are enormously useful in research as models of protein behaviour and as drug leads in therapeutics development.  This lecture will cover the way bioactive peptides are discovered and developed, including the way in which they are synthesised in the chemical laboratory.  Examples of their use in the design of new therapeutic agents will be described, including the search for new antimicrobial peptides and inhibitors of amyloid formation.

Protein production for biophysical characterisation
The preparation of recombinant proteins for research and development or for human therapeutic applications requires efficient procedures for the large-scale production of pure product.  The methods employed for such production and purification of protein, including recombinant protein bacterial or eukaryotic overexpression systems as well as chromatographic strategies, will be described. These technologies provide the platform for the biophysical characterisation of proteins and their interactions with other molecular species.

Redesigning proteins for improved human therapy
The introduction of recombinant DNA technology to the manufacture of therapeutic proteins has made possible the rational design and production of protein analogues with altered pharmacokinetic and pharmacological properties.  In this lecture, the structural approaches currently used in the design of recombinant proteins will be illustrated with human insulin, used in the management of diabetes mellitus and tissue-type plasminogen activator, used in the treatment of myocardial infarction.

PROTEIN FOLDING AND MISFOLDING IN DISEASE 

Concepts of protein folding
We will examine the implications of Levinthal’s Paradox which states that because there is an astronomical number of conformations open to the unfolded protein folding must proceed along defined pathways.  The different methods and techniques used to observe protein folding in the laboratory will also be discussed.

Mechanisms of protein folding
These lectures will review some of the current literature on the kinetics of protein folding, focusing on a comparison between small and large single domain proteins and the biological relevance of in vitro studies.  We will also examine how this information can be used to construct images of the folding process.

The polyglutamine repeat proteins and protein misfolding
Huntington’s disease is caused by the expansion of a polymorphic trinucleotide repeat (CAG that codes for glutamine) within exon 1 of the Huntington gene.  This expansion to greater than 40 repeats causes the protein which normally resides in the cytoplasm to translocate to the nucleus where it aggregates.  This lecture will examine the structural and biological consequences of glutamine expansion and describe the use of animal models in studying the disease.

The serpinopathies
The serpins represent a superfamily of proteins which undergo large structural changes.  However a number of human serpins are sensitive to mutations which destabilize the protein, causing aggregation and tissue deposition.  Using the serpin a₁-Antitrypsin as an example we will examine how mutations cause proteins to undergo conformational change and cause disease.

PROTEIN ENGINEERING

Overview of protein engineering and applications
With our current understanding of molecular biology and protein structure, it has become possible to redesign (engineer) proteins with desired functions.  In this lecture we will discuss both rational and combinatorial approaches to obtaining proteins with novel functions and we will explore a variety of applications of protein engineering.  Specific examples will be presented in detail in the following three lectures.

Orthogonal engineering of kinases for studying signaling pathways
Protein kinases (which catalyse protein phosphorylation reactions) play critical roles in numerous interconnecting signal transduction pathways.  Due to the complex networks of pathways, it has generally been difficult to identify the immediate targets of specific kinases.  Based on the known structures of kinases, it has become possible to engineer kinases that use a non-natural ATP analogue as the phosphate source.  These engineered kinases can now be used to identify the immediate targets of the corresponding natural kinases.

Engineering fluorescent proteins for cell biology
Certain organisms, such as jellyfish, naturally produce fluorescent proteins, which can be fused to natural proteins, allowing the natural proteins to be visualised in cells and tissues.  By protein engineering, mutants of natural fluorescent proteins have been developed with fluorescence signals at different wavelengths.  These different-coloured fluorescent proteins are powerful tools for monitoring protein expression, degradation, cellular localisation and protein-protein interactions in vivo.

Engineering the b-adrenergic receptor for structural determination
G protein-coupled receptors (GPCRs) play crucial roles in cellular communication and signalling and are the targets of numerous therapeutic agents.  However, until recently only one GPCR structure was known due to the difficulty purifying and crystallizing these integral membranes proteins.  By applying several clever protein-engineering tricks, it became possible to crystallize and solve the structure of the b-adrenergic receptor, a GPCR.  In this lecture we will discuss both the engineering approaches and the resulting structural insights.

BIOMOLECULAR INITERACTIONS & PROTEOMICS

Introduction to Proteomics
This lecture explores the relationship between the Proteome and the Genome and how the proteome can be studied experimentally.  The concepts of structural and functional proteomics will be introduced.  Introduction to proteomic methods of 2D gels, HPLC and mass spectrometry and their coupling with database search procedures to facilitate the bioinformatic identification.

Proteomic methods
This lecture discusses the integration of analytical techniques to construct he proteome of different organisms though the separation and identification of novel proteins, or new cellular pathways in normal cells.  Selected examples will also be discussed of the integration of proteomic technologies into medical bioinformatics in deciphering the molecular basis and mechanisms of disease and to increase opportunities to develop drugs with reduced side effects and in clinical diagnosis

Functional proteomics 
This lecture discusses the integration of analytical techniques to construct he proteome of different organisms though the separation and identification of novel proteins, or new cellular pathways in normal cells.  Selected examples will also be discussed of the integration of proteomic technologies into medical bioinformatics in deciphering the molecular basis and mechanisms of disease and to increase opportunities to develop drugs with reduced side effects and in clinical diagnosis

Protein-protein interactions 
Protein-protein interactions are the machinery that drive cellular function.  This lecture will cover biosensor techniques that are used to discover and characterise these interactions. The application of these techniques in the discovery of new pharmaceutical targets and drug design will be discussed.