Biophysics of Excitable Cells PHYS*2030 FALL 2002
Biological NanomaterialsNANO*4100 FALL 2014Instructor:John
DutcherOffice:MacN 451 Phone + phone mail : Ext.
53950E-mail:[email protected]
Web:www.physics.uoguelph.ca/psiLectures:M W F13:30 14:20MacN
201Course Website:
http://www.physics.uoguelph.ca/~dutcher/nano4100/
1Objectives of the CourseUnderstand the principles of the
quantitative biology approachUnderstand the basic building blocks
of biology and how they bind to form biological moleculesUnderstand
different interactions between biological molecules and the
principles underlying the self-assembly of aggregates of biological
molecules and nanomaterialsAppreciate the diversity and complexity
of self-assembled biological nanomaterialsExpand scientific writing
skills to develop effective communication
2LiteratureRequired Text: CD directory with review &
research papersAvailable in the cd directory at:
http://www.physics.uoguelph.ca/~dutcher/download/nano_4100Supplementary
Reading :Various journals related to biological molecules,
biological materials, nanomaterials (see the website for links)
Please learn how to use internet to look for papers and to find
their full texts. You should be familiar with the following: Entrez
(PubMed); ISI Web of Knowledge (Science Citation Index and
Biological Abstracts); Chemical Abstracts; Scholars Portal (or
ScienceDirect); HighWire Press; Annual Reviews; ACS
Publications3EvaluationProblem Assignments30% Directed Reading
Assignments 15% Marking of NANO*1000 Report 5% Midterm Test20%Final
Examination30%____________________________________Total100%
4Course Topics introduction to quantitative biology- power of
physical approach to biological systems introduction to
biomolecules and biological membranes- building blocks and
interactions lipids and self-assembly of lipid structures
macromolecules: polymers- random walks & diffusion
macromolecules: proteins & DNA- building blocks and higher
order structure self-assembly of macromolecules- copolymers,
protein filaments, peptide-based self-assembly biological machines-
bacterial flagella, myosin & kinesin walking, Brownian ratchet
bionanocomposites- unique properties5Guest InstructorsRob Wickham
(Physics):copolymersLeonid Brown (Physics):proteinsDoug Fudge
(MCB):protein filaments
6 liquid crystals surfactants colloids polymers biopolymers
cells foods
Soft Materials7Soft Materials bonding between molecules is weak
comparable to thermal energy kBT ~ 1/40 eV (@RT)
can have big changes to soft materials with small changes in
environment temperature, pH, ionic strength, applied fields8Soft
Materials hydrogelsC. Chang et al. Euro Polym J 46, 92 (2010)
Swollen in waterAs-preparedDriedSwollen in NaClsolution 9Soft
Materials rubber elasticityT. Russell, Science 297, 964 (2002)
StretchedUnstretched10Soft Materials drug delivery
heat-triggered dox release from Temperature Sensitive Liposome due
to MRI-guided high intensity focused ultrasound Grull &
Langereis, J Controlled Release 161, 317 (2012)
11Large Range of Length Scales properties depend on length scale
of measurement complex, hierarchical structure
processing is the key[P. Ball, Made to Measure]12Physics Meets
Biology bring together biology & physics to get biological
physics sophisticated experimental tools sophisticated models of
biological systems
Quantitative Biology quantitative data demand quantitative
models www.qbio.ca
13PSI Biological Physics Projects bacterial biophysics
viscoelastic properties of bacterial cells bacterial twitching
motility Min protein oscillations & patterns
biopolymers at surfaces & membranes single molecule pulling
of proteins on nano-curved surfaces single molecule imaging of
peptides in lipid matrix field driven changes in conformation &
orientation
enzymatic degradation of cellulose imaging & kinetics of
adsorption & degradation
polysaccharide nanoparticles startup company
14Quantitative Biology eight fundamental concepts provide
toolbox for interpreting biological data simple harmonic oscillator
ideal gas & ideal solutions Ising model random walks, entropy
& diffusion Poisson-Boltzmann model of charges in solution
elastic theory of 1D rods & 2D sheets Newtonian fluid model
& Navier-Stokes equations rate equation models of chemical
kineticsAdapted from Phillips et al., Physical Biology of the
Cell15Quantitative Biology simple harmonic oscillatorPhillips et
al., Physical Biology of the Cell
16Quantitative Biology different levels of modeling beyond the
spherical cowPhillips et al., Physical Biology of the Cell
DNA
membrane
17Rules of ThumbPhillips et al., Physical Biology of the
Cell
18Rules of ThumbPhillips et al., Physical Biology of the
Cell
19Drunkards walk
Courtesy of GeorgeGamowRandom Walks
Random Walk Common Theme random walk is a recurring concept in
course helps with seemingly unrelated problems diffusion of
molecules, cells & nanomachines polymer conformation protein
conformation compact random walk other non-obvious implementations
packing of chromosomes in nuclei looping of DNA fragments DNA
melting molecular motors21
N = 1000
Gaussian random walk
(b) self-avoiding random walkPolymer ConformationabRandom coils
are loosely-packed structures22
Self-Similarity of a Polymer Molecule
23Swimming of Bacteria
Contribution of Physical Science to Biology Is Hard to
OverestimateX-ray
NMRESREMPDERGS9-1Gt/i1-1.5+1.5-5.5+5.5ppm (1H)ppm (13C)from
Ridge et al.Made it to here after first lecture F1225Case Study of
Bacteriorhodopsin - Contribution of Physical Methods
from Luecke et al. 7 transmembrane helices light-driven ion
pumpYoutube video on bacteriorhodopsinfrom Alberts et al.26Case
Study of Bacteriorhodopsin - Contribution of Physical MethodsUV/Vis
spectroscopy - kinetics and thermodynamics of the photocycle,
orientation of the chromophore (LD)Raman spectroscopy -
configuration of the retinal chromophore and its changes in the
photocycleFTIR spectroscopy - conformational changes of the protein
and its chromophore in the photocycle, protonation changes of
carboxylic acidsNMR spectroscopy - structure of protein fragments,
orientation of the chromophore, dynamics of certain residues ESR
spectroscopy - protein topology, conformational changesElectron,
Neutron, X-ray diffraction - structure of the protein and its
intermediates, location of water moleculesAtomic force microscopy -
single molecule imaging & spectroscopyQuantum
chemistry/Molecular Dynamics - properties of the chromophore and
its binding site27CellsMany different kinds of cellsProkaryotic
cellsRelatively simple membrane structureFew internal
membranesEukaryotic cellsPlant cellsPlasma membrane inside the cell
wallInternal chloroplastsAnimal cellsPlasma membraneNuclear
membrane
28Dynamics of Cells
Youtube video on the Inner Life of the Cellfrom Biovisions
project @ HarvardSwimming bacteria (Howard Berg)Pilus retraction
(Howard Berg)
29Biological MembranesMajor functions of cell membranes:To
separate interior and exterior of the cellTo maintain concentration
gradients of various ions, which serve both as sources of energy
and as a basis for excitabilityTo house functionally important
protein complexes such as energy-producing machines, transporters,
enzymes, and receptors
From Lodish et al30Biological MembranesCryo-electron microscopy
reveals detailed structurePhillips
V. Matias, U of GuelphPhD thesis C. crescentusIntestinal
epithelial cellsPhotoreceptors in rod cellMitochondrian surrounded
by endoplasmic reticulumS. aureus septum
31Major Components of a Membrane
MembraneProteinsLipid Bilayer Characteristic molecular
weightsLipids: 0.5-2 kDaProteins: 5-6000 kDafrom Luecke et al.Other
components: carbohydrates, water, ionsDalton: unified atomic mass
unit (amu), 1 g/mol, mass of one nucleon32Fluid Mosaic Model
From CooperSinger & Nicolson, Science (1972)33Evolution of
Membrane ModelsPhillips, Physical Biology of the CellSackmann
(1995)
Singer & Nicolson (1972)
Israelachvili (1978)
34Restrictions to Free Diffusion of Membrane Proteins
from Vereb et al.A lipid microdomainsB, C cytoskeletonD protein
association35Hydration of a Lipid Bilayer (MD Simulation)
from Popot and Engelman36
Membrane Proteins and Lipids Are Often Linked with Carbohydrates
(glycoproteins and glycolipids)From Lodish et al37Building a Lipid
Molecule Start with fat Long chain hydrocarbon Different numbers of
carbons with either Single bonds (saturated) Double bonds
(unsaturated)
Convert hydrocarbon chain to fatty acid by attaching carboxyl
(-COOH) group at end Fatty acids are fundamental building block of
lipids 2 to 36 carbons long, with most common between 14 & 22
Usually even number of carbons most fatty acid chains are
unsaturated single double bond most common, up to 6 double
bonds
e.g. oleic acide.g. DHA (docosahexaenoic acid)38Building a Lipid
Molecule fatty acids rarely found free in cell chemical linking to
hydrophobic group, e.g. glycerol, produces non-polar lipid
di-acylglycerol has 2 fatty acids Key lipid in signaling pathways
tri-acylglycerol is typical storage fat can replace one of the
fatty acids with a polar group polar lipid or glycero-phospholipid
hydrophobic tail & hydrophilic head e.g. PC, PE, PG, PI
PC: phosphatidylcholine or lecithin PE: phosphatidylethanolamine
PG: phosphatidylglycerol PI:
phosphatidylinositolneutralcharged39Building a Lipid Molecule
polarhydrophobicFatty acid myristic acid (14:0)Oleic acid
(18:1)DHA (22:6)Di-acylglycerol of myristic acidTri-acylglycerol of
stearic acid(triglyceride)glycerolFrom Mouritsen40Building a Lipid
MoleculepolarhydrophobicDMPC lipid:di-acylglycerol
&phosphatidylcholinelysolipidPhosphatic acid
phosphateglycerolcholineFrom Mouritsen41Phospholipids: Structure
Overview
Typical PhospholipidAmphipathic Nature!Polar,
HydrophilicNon-Polar, HydrophobicVariableFrom Renninger42Major
Phospholipids
From Alberts et alglycerolphosphatecholine43Major
PhospholipidsFrom Mouritsen
Made it to here after second lecture F1244Major
PhospholipidsFrom Mouritsen
45Glyco(sphingo)lipids
From Alberts et al46Cholesterol Stiffens Fluid Membranes
From Alberts et al
47Lipid Rafts
From Dykstra et al48Phase Transitions in Lipid Layers Can use
differential scanning calorimetry (DSC) Heat sample and reference
(material similar to sample but not does have phase transition in
the region of interest) at identical rate e.g. sample is lipid +
solvent, reference is solvent At phase transition, more heat must
be applied to the sample to maintain the linear increase in
temperature with time The excess or differential heat supplied to
the sample is recorded as a function of temperature The sensitivity
depends on the sample size, but also on scan rate At a phase
transition, get a peakTm: peak position (phase transition
temperature)DT1/2: FWHM of peakDH: area under the peak (enthalpy of
transition)DS = DH/Tm: entropy of transition49Differential Scanning
Calorimetry
variation of excess specific heat with temperature for
two-state, endothermic process50Differential Scanning
Calorimetry
51Differential Scanning Calorimetry
52Differential Scanning Calorimetry
DSC curves of distearoyl PC (DSPC) layers as a functionof water
content CChapman et al., Chem. Phys. Lipids (1967)Peak at 62 deg
C53Lipid Layer OrderingShort range order described bya: chains are
disordered (melted) Trans-gauche isomerization Rapid diffusion
(translation & rotation)b: chains stiff, oriented parallel to
each other, perpendicular to bilayer planeb: chains tilted with
respect to bilayer normalc: crystalline phase (Lc is lamellar but
crystalline within the plane)
Long range order described byL: 1D lamellarT: 3D tetragonalP: 2D
rectangularR: rhombohedralH: 2D hexagonalQ: cubic54Lipid Layer
Ordering
55Lipid Phase Diagram
Blume, Acta ThermChimActa (1991)Phase diagram for PC/water
systems56Lipid Phase Transition Gel to liquid crystal phase
transition involves
Cooperative melting of hydrocarbon chains Introduces large
number of trans-gauche isomerizations Introduces kinks and jogs
into chains
Large increase in lateral diffusion rate of lipids in plane of
bilayer
Small increase in volume Large increase in area per polar head
Decrease in bilayer thickness
Observed not only in model systems but also in whole cells
57Lipid Phase Transitions Can investigate changes in transition
temps with chain length, etc.
Blume, Acta ThermChimActa (1991)58Lipid Phase Transitions
Blume, Acta ThermChimActa (1991)Dependence of DH and Tmon
position of double bondin PCs with chain length of18 carbons
Nature can control Tm byplacement of double bond59Influence of
Polar Head Group PEs have a higher Tm than PCs smaller headgroup
for PE hydrogen bonding of PE protonated amino group with adjacent
negatively charged phosphate group note effect of pH increase pH to
12 to deprotonate PE headgroup Tm decreases from 63oC to 41oC for
DPPE
PG negatively charged in high ionic strength solvent, charges
are shielded at neutral pH, Tm, DH and DS for PGs are similar to
those for PCs
PS at neutral pH, 2 negative charges and 1 positive charge Tm
influenced by pH and ionic strength
60Lipid MonolayersNot a bilayer, but
Well defined geometry with which to study the intermolecular
interactions between lipids and between lipids & proteins
Create a so-called Langmuir monolayer by spreading amphiphilic
molecules at the air-water interface using a Langmuir trough
Movable barriers allow the control of the surface area A which
causes a change in the surface pressure p
This allows measurement of the p-A isotherm, which has
characteristic shape for each type of molecule and provides
information about the orientation and packing of the molecules
61Langmuir TroughNorde, Colloids and Interfaces in Life Sciences
(2003)Schematic of Langmuir trough
62Surface Pressure-Area IsothermNorde, Colloids and Interfaces
in Life Sciences (2003)G: gas; LE: liquid expanded; LC: liquid
condensed; S: solid
63Phase CoexistenceNorde, Colloids and Interfaces in Life
Sciences (2003)Brewster angle microscopy of monolayers showing
theCoexistence of LC (light) and LE (dark) phases
64Compressibility slope of p-A isotherm is measure of isothermal
compressibility
monolayer in gas state is highly compressible but it is less in
more condensed states
65Phase CoexistenceNorde, Colloids and Interfaces in Life
Sciences (2003)
Orientations of amphiphilic moleculesfor the various phaseson
the pressure-areaisotherms66Temperature Dependence of p-A
IsothermsNorde, Colloids and Interfaces in Life Sciences (2003)
as temperature increases pressure at onset of LE LC transition
increases corresponding value of am decreases coexistence region
decreases67Albrecht et al., J. Phys. (Paris) (1978)p-A isotherms
for DPPC at different temperatures
Temperature Dependence of p-A Isotherms68
Langmuir-Blodgett Film FormationNorde, Colloids and Interfaces
in Life Sciences (2003) formation of Y-type Langmuir-Blodgett film
transfer rates of ~1 mm/s69Langmuir-Blodgett Film FormationNorde,
Colloids and Interfaces in Life Sciences (2003) X-type transfer
Z-type transfer can also use Langmuir-Schaefer deposition
horizontal touch of substrate on monolayer70