Biophysics: From Proteins to Cells

January 15-26, 2001, Canberra, Australia

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Lecture Topics & Abstracts

Robert Austin (Physics, Princeton)

Energy and population landscapes in biology: a physics perspective

Biology "In Silico": Interfacing biology to the microfabricated world Silicon micromachining has opened up a new world of sub micron spaces in which you can study biological objects on a scale commensurate with their size and operational environment. Since silicon micromachining is so highly sophisticated in terms of technology, it is possible to design and construct highly creative structures which can probe specific aspects of a biological object. Such structures can also be very practical and useful in applied areas such as biotechnology.

The manipulation and sorting of biological particles poses unique challenges to microfabrication because of the complex physical properties of biological particles. These properties range from size (DNA is an extremely long but thin polymer while the cell is a compact sphere) to adhesive properties (white blood cells are selectively extremely sticky while red blood cells are designed to be quite non adhesive). An even more important issue is the fact that each biological particle, whether it be the sequence of a DNA fragment or a white blood cell, is unique. Often it is vital to ascertain the uniqueness of the particles, to sort them and find a very rare individual in a population of millions. I will present examples of our attempts to attack.

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Avinoam Ben-Shaul (Physical Chemistry, Hebrew University)

Interaction mechanisms and phase transitions in membrane-macromolecule systems

The lipid-protein membrane, defining the boundary between the inside and outside of cells and cell organells, is a self-assembled multicomponent system of remarkable physico-chemical characteristics. Its central structural element, the lipid bilayer, is an elastic membrane capable of undergoing bending and stretching deformations without losing its integrity. Also, within the membrane plane lipids and proteins can diffuse laterally, implying that the membrane is, in fact, a two-dimensional fluid mixture. The elastic and compositional (lateral diffusion) degrees of freedom of lipid membranes play a crucial role in their interactions with peripheral or integral macromolecules such as adsorbed or embedded proteins. For instance, an integral hydrophobic protein can induce local changes in the curvature, thickness and composition of its lipid surroundings. Such membrane perturbation can result in protein aggregation or even morphological phase transitions of the lipid-protein matrix. The lectures presented in this summer school will review some of the basic physico-chemical properties of lipid membranes and their interactions with integral (hydrophobic) proteins and peripheral (electrically charged) molecules, such as DNA and basic proteins. The emphasis will be on a molecular-level theoretical treatment of these interactions and simple statistical-thermodynamic analysis of their consequences. More specifically, the four lectures will focus on the following topics.

Lecture 1. The lipid bilayer.

The integrity, fluidity and elasticity of lipid membranes are intimately related, and determined, by the amphiphilic nature of their constituent molecules. The basic forces and principles governing the molecular organization of lipid membranes will be reviewed, emphasizing the important role of amphiphile chain conformational statistices. Some relevant molecular-thermodynamic quantities, such as the lateral pressure profile and chain orientational order, will be introduced and related to the membrane free energy, elastic muduli and spontaneous curvature.

Lecture 2. Hydrophobic proteins in membranes.

An integral membrane protein "perturbs" the organization of the lipid molecules in its vicinity. The perturbation free energy depends on the shape of the protein, the nature and composition of the lipid mixture and the "hydrophobic mismatch" (i.e., the difference between the length of the protein and the thickness of the protein-free membrane). The elastic deformation of the membrane can result in 'lipid-mediated' interactions between embedded proteins which may be repulsive or attractive, depending on the structural characteristics of the membrane and the proteins. In certain cases the lipid-mediated interaction can lead to protein aggregation or to more dramatic effects such as structural phase transitions of the lipid-protein matrix. The lecture will review the current theoretical understanding of these phenomena.

Lecture 3. Electrostatically adsorbed proteins.

Charged proteins are attracted to oppositely charged membranes. The major driving force for this attraction is the entropy gain associated with the release of the mobile counterions (which surround the separated protein and membrane) into the bulk solution. The adsorption energy depends on the charge distributions on the protein and membrane surfaces. Biological membranes are generally weakly charged, since only a small fraction of their constituent lipids carry charged head groups. However, because the lipids are mobile in the membrane plane, charged lipids can diffuse towards (or, rarely, awayfrom) the approaching protein, thus enhancing protein adsorption. This, in turn, results in unfavorable "lipid-demixing" and the appearance of composition gradients in the membrane. These, as well as lateral interactions between the adsorbed proteins determine the adsorption characteristics of the protein overlayer. In certain cases, the interplay between the attractive and repulsive contributions to the adsorption free energy can lead to two-dimensional phase transitions. The molecular mechanisms underlying these phenomena will be described based on familiar statistical-thermodynamic approaches, such as the Poisson-Boltzmann theory for the electrostatic interactions.

Lecture 4. DNA-lipid complexes.

The mixing of aqueous solutions of DNA and liposomes containg cationic lipids results in the spontaneous formation of periodic composite aggregates containg lipid layers and DNA. These, typically micron size, complexes are considered as promising DNA delievery vectors for gene therapy, in which context they are often referred to as "lipoplexes". Their formation is driven by the electrostatic attraction (counterion release...) between the negatively charged DNA and the positively charged membranes. The structure of these complexes is determined by a delicate balance between electrostatic forces, the chemical composition of the lipid mixture and by the elastic properties of the lipid layers, primarily by their bending rigidity and spontaneous curvature. Both lamellar complexes (consisting of stacks of lipid bilayers and DNA strands sandwiched between them) and hexagonal complexes (consisting of DNA strands intercalated within the water tubes of an inverse-hexagonal lipid matrix) have been observed and quantitaively characterized. Phase transitions between these structures can be mediated by changing the lipid mixture or the DNA/lipid concentration ratio. The structure, stability and phase behavior of these recently discovered systems will be interpreted based on principles and ideas introduced in previous lectures.

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Ken Dill (Pharmaceutical Chemistry, UC - San Francisco)

The statistical mechanics of protein folding

Lecture 1: Polymer principles applied to protein folding.
Lecture 2: Water and the hydrophobic effect.
Lecture 3: Protein folding energy landscapes, and protein structure prediction.
Lecture 4: Modeling conformational transitions in proteins and RNA molecules.

Statistical mechanical models have been developed in recent years for the structures, thermodynamics, and kinetics of protein folding. The aim has been a better understanding of the full conformational spaces of proteins, entropies of folding, non-native conformations such as transition states, intermediates, molten globules, and the conformational transitions between them. Such states can be described in terms of free energy landscapes. Models of landscapes have begun to be useful for developing faster conformational search methods that
can help predict native protein structures from their amino acid sequences. To further improve the predictive power of computational models for proteins and RNA molecules also requires a better understanding of aqueous solvation. For these processes, too, there are some new results from statistical mechanical modeling.

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Peter Kollman (Pharmaceutical Chemistry, UC - San Francisco)

Structures and free energies of proteins and nucleic acids from molecular dynamics simulations

We will give a description of how molecular dynamics simulations can be carried out for macromolecules, in order to use this methodology in
studies of protein folding, protein structure prediction, ligand binding and enzyme action. Our four lectures will be divided into the following topics:

References:
1. Cornell et al, JACS, 117, 5179(1995)
2. Kollman, Chem. Rev., 93,2395(1993)
3. A. Leach,Molecular Modeling:Principles and Applications, Longman,1996

4. Massova and Kollman, JACS, 121,8133(1999)
5. Stanton et al, JACS, 120,3448(1998)

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Jack Tuszynski (Physics, Alberta)

Models of the collective behavior of proteins in cells: actin, tubulin and motor proteins

Lecture 1: Molecular dynamics studies of tubulin and their significance

Nogales et al have recently reported the structure of alpha- and beta-tubulin based on their atomic resolution crustallography work. We used the publically available data from the PDB and studied a number of physical characteristics of the tubulin heterodimer. First, using the TINKER package we computed the overall charge and dipole moment on the tubulin molecule. The value of the charge must be corrected for the C-terminus contribution which has not been imaged by Nogales et al. The results we obtained for the dipole moment indicate the existence of a large net dipole (on the order of 1700 Debye) whose protofilament component is in agreement with our earlier estimates. Finally, we have carried out calculations of the electrostatic potential around a micrtubule at various distances from the surface and at different locations with respect to the surface. We have found profiles which are qualitatively consistent with earlier theoretical predictions. The new quantitative aspects of our calculations shed light on the possible mechanism of motor protein motion along the microtubule. This is work in progress and many questions remain to be answered in regard to the effect of various isotypes, the inclusion of the C-terminus, the conformational changes affected by the GTP hydrolysis and binding of ligands and drug molecules to the tubulin surface.

Lecture 2: Models of polymerization kinetics for microtubules and actin filaments: from linear kinetics to collective synchronized oscillations.

This lecture provides an overview of the commonly used approaches in the description of biopolymer filament formation. We first discuss linear polymerization kinetics of actin filaments and the interesting extensions used to model the emergence of actin bundles. We then turn to the description of the evolution of microtubule lengths at both low- and high-density concentrations of tubulin. We derive general master-type equations which are based on the key chemical reactions involved in the assembly and disassembly of microtubules. The processes included are: polymerization and depolymerization of a single protein dimer, catastrophic disassembly affecting an $\acute{a}$ piori arbitrary number of dimers, and a rescue event. Solutions of the derived equations are compared with the existing experimental data. Important conclusions linking the emergence of bell-shaped histograms with the nature of catastrophe and rescue phenomena are drawn. Finally, we briefly discuss the emergence of coherent phenomena in microtubule polymerization, i.e. a transition to collective oscillations in the assembly and disassembly effects.

Lecture 3: Model of motor protein motion along microtubule filaments.

In this talk I'll first discuss the key biophysical properties of motor proteins which have become a target of intensive investigations in cell biology, biochemistry and related fields due to the recognition of their importance in subcellular transport processes. There has also been a surge of interest intended to provide mathematical and physical models of motor protein motion along biopolymer filaments in the cell which will be briefly described. Models for molecular motors in ratchet potentials in terms of Fokker-Planck equations, Newtonian particles with friction and master equations will be contrasted and compared. I will then outline a simple phenomenological model for motor protein motion along microtubule protofilaments. The model assumes the presence of an effective potential due to electrostatic effects which has a different character in the direction parallel to the protofilament from the direction perpendicular to the protofilament axis. In the direction perpendicular to the axis, the potential is an asymmetric double well with an overall minimum at the filament surface and a metastable local minimum a few nanometers away from it. The latter effect can be removed if the average concentration of ATP in the solution drops below a critical value. The relative position of the minima depends on the value of ATP concentration which affects the relaxation time and hence the average velocity of motor protein motion. Parallel to the filament axis, the effective potential is periodic with a constant tilt due to a net electric field effect. It resembles the so-called washboard potential frequently used in the physics of semiconductors and superconductors. I discuss the physical mechanisms involved in each of the steps of motor protein motion and derive requisite differential equations of motion for the motor protein. Finally, I compare our results with a number of experimental data such as the dependence of the average velocity on the ATP concentration, the effects of externally applied electric fields and temperature. I also examine the effects of discreteness on the requisite differential equations of motion with both mathematical and biophysical consequences.

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David Adams (Biomedicine, Queensland)

Expression and function of membrane ion channels

Lecture 1
Methods to study ion channel expression and function. Use of molecular
biological and electrophysiological techniques to provide information
about the structure and function of ion channels. Techniques used to
measure electrical activity in excitable cells - patch clamp recording
configurations to monitor and analyze ion channel function. Analysis of
single channel records.

Lecture 2
Voltage-gated ion channels: Na+, Ca2+, K+ channels. Distribution and
function of voltage-gated ion channels in neurotransmission. Molecular
structure, gating and selectivity of voltage-gated ion channels.
Modulation of voltage-gated ion channels by neurotransmitters and G
proteins. Mechanisms of block of ion channels by drugs and toxins.

Lecture 3
Ligand-gated ion channels: nicotinic ACh receptor and other
neurotransmitter-gated ion channels.
G protein-gated ion channels. Receptor-channel expression and
localization. Molecular structure, gating and selectivity of
ligand-gated ion channels. Second messengers and role of protein
phosphorlyation in modulation of ion channel function and synaptic
transmission.

References :
1. D.J. Aidley and P.R. Stanfield (1996) Ion Channels. Molecules in
Action. Cambridge University Press.
2. F.M. Ashcroft (2000) Ion Channels and Disease. Academic Press, San
Diego, CA
3. P.M. Conn (1998) Ion Channels. Vols. 293, 294. Methods in Enzymology.
Academic Press, San Diego, CA
4. B. Hille (1992) Ionic Channels of Excitable Membranes. 2nd Edition.
Sinauer Associates Inc., Sunderland, MA
5. D. Ogden (1994) Microelectrode Techniques. The Plymouth Workshop
Handbook. 2nd Edition. The Company of Biologists Ltd., Cambridge.
6. B. Sakmann and E. Neher (1995) Single-Channel Recording. 2nd Edition.
Plenum Press, New York.

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Shin-Ho Chung (Chemistry, Faculties, ANU)

Theories of ion permeation in membrane channels

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Hans Coster (Biophysics, UNSW, Sydney)

Physics of cell membranes

The structure of cell membranes
Self assembly and molecular organisation of membranes
Dielectric and Electrical properties of cell membranes
Biotechnology applications founded on membrane physics

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Paul Gooley (Biochemistry, Melbourne)

Analysis of proteins and their complexes by nuclear magnetic resonance

Lecture 1: Determining the structure of a protein by heteronuclear NMR
spectroscopy

Lecture 2: Using X-filtered experiments to determine the structure of
protein complexes.

Lecture 3: New strategies for determining the structure of large proteins
and improving the accuracy of a structure.

The solution of small to medium sized proteins by heteronuclear NMR spectroscopy has become a routine and established method. In these lectures I will describe the application of three and four dimensional triple resonance and heteronuclear edited experiments for the assignment of NMR spectra and the strategies for determining the structure of a protein. Particular emphasis will be given to determining the structures of protein complexes and the different experimental approaches used for weak to strong binding conditions. Recently developed methods for elucidating the structure of large proteins and for improving the accuracy of a structure will also be discussed.

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Michael Parker (St. Vincent's, Melbourne)

Protein structure from x-ray diffraction

Protein crystallography is the study of the three-dimensional structures of proteins at near atomic resolution. The field has provided a tremendous insight into numerous biological processes since the first structures were revealed in the 1960’s. The field has undergone a massive worldwide expansion in the last decade, not only in academic labs but also in the pharmaceutical industry. The main driving force for this expansion has been the use of the three-dimensional atomic structures of proteins to design drugs for a variety of diseases. In these lectures I will describe the pathway involved in going from protein to structure using the technique of X-ray diffraction.

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Ron Pace (Chemistry, Faculties, ANU)

Biosensors

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Tim Senden (Applied Maths, RSPS&E, ANU)

Atomic force microscopy

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Edith Sevick (RS Chemistry, ANU)

Optical tweezers

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Fred Chow (Photobioenergetics, RSBS, ANU)

Photosynthetic systems

Oxygenic photosynthesis has evolved over three billion years,providing food, fiber and fuel for the biosphere. The natural process comprises the absorption of light and transfer of excitation energy among an array of light-harvesting pigments towards a special reaction center. At this center the primary charge separation takes place, occurring with a quantum efficiency of Š 95%. The separated electron and hole is then stabilized by secondary electron transfers over a series of intermediates and results in the splitting of water intooxygen gas, electrons and protons. The free energy released is subsequently stored in the form of carbohydrates and other organic compounds.

In recent years an increasing effort has been made to mimic aspects of natural photosynthesis in artificial systems. These include lightharvesting in multi-chromophoric arrays, charge separation in covalently-linked electron donor/acceptor pairs, and redox reactions in synthetic polypeptides. In this lecture, the basic principles of photosynthetic energy conversion will be described and examples of artificial systems will be presented to illustrate the potential developments in this field of biotechnology.

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Date last modified: 6 July 2000
Mail problems to adm105@rsphysse.anu.edu.au
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