[toc]At the end of this course you will have an understanding of:
- the hydrophobic effect and how it leads to deformable membranes in which proteins can function.
- facilitated transport across membranes, the structure of ion channels, and how the latter have exploited energetic principles found in simple ionophores to achieve transport rates of close to theoretical maximum.
- how active transport against a concentration gradient is achieved using Bacteriorhodopsin and Ca-ATPase as prototypal examples. How pumps differ from channels.
- pKa, how an ion pump might utilise changes in side chain pKa to achieve vectorial ion translocation through the protein, and the nature of electrostatics within a protein interior where the dielectric is low.
- the nature of ATP sites in proteins, the extent and nature of domain movements, chemo-mechanical coupling.
This module descriptor document also can be downloaded as an MS-Word document.
Deputy Programme Co-ordinator, Associate Professor David McIntosch, from Chemical Pathology, will co-ordinate the programme at UCT’s Institute of Infectious Disease and Molecular Medicine (IIDMM). He will also present a workshop on the special problems associated with the structure determination of membrane proteins as well as a series of lectures in which major achievements of structural biology are reviewed in context.
- Forces contributing to protein stability, flexibility, domain dynamics
- The hydrophobic effect
- Electrostatic interactions
- Hydrogen bonding
- Van der Waals interactions
- Binding energy
- Chemical energy from group transfer
- Electro-chemical potential
- Bacteriorhodopsin – the prototypal membrane H+ pump powered by light
- The catalytic cycle and conductance pathway
- Mechanism of pKa changes
- Calculating internal pKa’s using a Finite Difference Poisson-Boltzmann method
- Conformational shifts, energy coupling, and vectoriality, according to crystal structures of four key catalytic intermediates.
- Ca-ATPase – an electogenic ion pump powered by ATP
- Catalytic cycle defines the key intermediates
- A crystal structure of one conformation suggests long-range energy coupling and large domain movements during pumping
- Electrostatic fireworks in the engine room deduced from site-directed mutagenesis
- Possible mechano-coupling models
- FoF1-ATP synthase-a rotary H+ pump powered by ATP
- Rotary catalysis linked to binding changes (rather than ATP hydrolysis) was predicted from oxygen Pi-HOH exchanges and kinetics
- Proof of rotary movement from crystal structure, cross-linking, and single molecule dynamics
- Turning an electrochemical gradient into rotary movement
- Myosins and kinesins – motors powered by ATP to run along tracks
- Methods for detecting bending movements
- Determinants of motor directionality
- Force and velocity measurements of single molecules
There will be two assignments:
- Detail the nature of the forces that a K+ ion is subjected to as it traverses a K+ channel.
- Describe the path in atomic detail that a H+ follows during passage through bacteriorhodopsin. Provide reasons at each critical point why it goes in an extracellular direction rather than intracellular.
The project could involve expression of an ion pump from Mycobacterium tuberculosum, or a soluble portion thereof, in preparation for crystalization or NMR structure determination.
David McIntosch: Physical Methods For Probing Membrane Protein Structure And Biological Function
/mcintosh/Lecture4_Oct22.ppt on one of the servers.
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