Biological Nanomaterials
NANO*4100 FALL 2014
Lectures:
MWF
Instructor:
Office:
Phone + phone mail :
E-mail:
Web:
13:30 – 14:20
MacN 201
John Dutcher
MacN 451
Ext. 53950
dutcher@uoguelph.ca
www.physics.uoguelph.ca/psi
Course Website:
http://www.physics.uoguelph.ca/~dutcher/nano4100/
Objectives of the Course
• Understand the principles of the quantitative biology
approach
• Understand the basic building blocks of biology and how
they bind to form biological molecules
• Understand different interactions between biological
molecules and the principles underlying the selfassembly of aggregates of biological molecules and
nanomaterials
• Appreciate the diversity and complexity of selfassembled biological nanomaterials
• Expand scientific writing skills to develop
effective communication
Literature
Required Text:
“CD” directory with review & research papers
Available in the “cd” directory at:
http://www.physics.uoguelph.ca/~dutcher/download/nano_4100
Supplementary 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 Publications
Evaluation
Problem Assignments
30%
Directed Reading Assignments
15%
Marking of NANO*1000 Report
5%
Midterm Test
20%
Final Examination
30%
____________________________________
Total
100%
Course 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 properties
Guest Instructors
• Rob Wickham (Physics):
• Leonid Brown (Physics):
• Doug Fudge (MCB):
copolymers
proteins
protein filaments
Soft Materials
• liquid crystals
• surfactants
• colloids
• polymers
• biopolymers
• cells
• foods
Soft 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 fields
Soft Materials
• hydrogels
As-prepared
Swollen in water
Dried
Swollen in NaCl
solution
C. Chang et al. Euro Polym J 46, 92 (2010)
Soft Materials
• rubber elasticity
Stretched
Unstretched
T. Russell, Science 297, 964 (2002)
Soft 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)
Large Range of Length Scales
• properties depend on length scale of measurement
− complex, hierarchical structure
processing is the key
[P. Ball, Made to Measure]
Physics 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
PSI 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
Quantitative 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 kinetics
Adapted from
Phillips et al., Physical Biology of the Cell
Quantitative Biology
• simple harmonic oscillator
Phillips et al., Physical Biology of the Cell
Quantitative Biology
• different levels of modeling
• beyond the spherical cow
membrane
DNA
Phillips et al., Physical Biology of the Cell
Rules of Thumb
Phillips et al., Physical Biology of the Cell
Rules of Thumb
Phillips et al., Physical Biology of the Cell
Random Walks
Drunkard’s walk
Courtesy of George
Gamow
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 motors
Polymer Conformation
a
N = 1000
(a) Gaussian
random walk
b
(b) self-avoiding
random walk
Self-Similarity of a Polymer Molecule
Swimming of Bacteria
Contribution of Physical Science to
Biology Is Hard to Overestimate
PDE
RGS9-1
from Ridge et al.
X-ray
NMR
-1.5
+1.5
ppm
(1H)
ppm
(13C)
-5.5
Gt/i1
EM
+5.5
ESR
Case Study of
Bacteriorhodopsin Contribution of
Physical Methods
• 7 transmembrane
helices
• light-driven ion pump
Youtube video on bacteriorhodopsin
from Alberts et al.
from Luecke et al.
Case Study of Bacteriorhodopsin - Contribution of
Physical Methods
• UV/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 photocycle
• FTIR spectroscopy - conformational changes of the protein and its
chromophore in the photocycle, protonation changes of carboxylic
acids
• NMR spectroscopy - structure of protein fragments, orientation of the
chromophore, dynamics of certain residues
• ESR spectroscopy - protein topology, conformational changes
• Electron, Neutron, X-ray diffraction - structure of the protein and its
intermediates, location of water molecules
• Atomic force microscopy - single molecule imaging & spectroscopy
• Quantum chemistry/Molecular Dynamics - properties of the
chromophore and its binding site
Cells
Many different kinds of cells
• Prokaryotic cells
• Relatively simple membrane structure
• Few internal membranes
• Eukaryotic cells
• Plant cells
• Plasma membrane inside
the cell wall
• Internal chloroplasts
• Animal cells
• Plasma membrane
• Nuclear membrane
Dynamics of Cells
Swimming bacteria (Howard Berg)
Pilus retraction
(Howard Berg)
Youtube video on the Inner Life of the Cell
from Biovisions project @ Harvard
Biological Membranes
Major functions of cell membranes:
1. To separate interior and exterior of the cell
2. To maintain concentration gradients of various ions,
which serve both as sources of energy and as a basis
for excitability
3. To house functionally important protein complexes
such as energy-producing machines, transporters,
enzymes, and receptors
From Lodish et al
Biological Membranes
Cryo-electron microscopy reveals detailed structure
(A) C. crescentus
(B) Intestinal epithelial cells
(C) Photoreceptors in rod cell
(D) Mitochondrian surrounded
by endoplasmic reticulum
Phillips
S. aureus septum
V. Matias, U of Guelph
PhD thesis
Major Components of a Membrane
from Luecke et al.
Membrane
Proteins
Characteristic molecular weights
Lipids: 0.5-2 kDa
Proteins: 5-6000 kDa
Lipid
Bilayer
Other components: carbohydrates, water, ions
Fluid Mosaic Model
Singer & Nicolson, Science (1972)
From Cooper
Evolution of Membrane Models
Singer & Nicolson (1972)
Israelachvili (1978)
Phillips, Physical Biology of the Cell
Sackmann (1995)
Restrictions to Free Diffusion of Membrane
Proteins
A – lipid microdomains
B, C – cytoskeleton
D – protein association
from Vereb et al.
Hydration of a
Lipid Bilayer
(MD
Simulation)
from Popot and Engelman
Membrane Proteins and Lipids Are Often Linked
with Carbohydrates (glycoproteins and
glycolipids)
From Lodish et al
Building 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 acid
e.g. DHA (docosahexaenoic acid)
Building 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
neutral
charged
• PC: phosphatidylcholine or lecithin
• PE: phosphatidylethanolamine
• PG: phosphatidylglycerol
• PI: phosphatidylinositol
Building a Lipid Molecule
polar
hydrophobic
Fatty acid myristic acid (14:0)
Oleic acid (18:1)
DHA (22:6)
Di-acylglycerol of myristic acid
Tri-acylglycerol of stearic acid
(triglyceride)
glycerol
From Mouritsen
Building a Lipid Molecule
polar
hydrophobic
glycerol
choline
DMPC lipid:
di-acylglycerol &
phosphatidylcholine
phosphate
lysolipid
Phosphatic acid
From Mouritsen
Phospholipids: Structure Overview
Amphipathic Nature!
Polar, Hydrophilic
Non-Polar, Hydrophobic
Variable
Typical Phospholipid
From Renninger
Major Phospholipids
From Alberts et al
choline
phosphate
glycerol
Major Phospholipids
From Mouritsen
Major Phospholipids
From Mouritsen
Glyco(sphingo)lipids
From Alberts et al
Cholesterol “Stiffens”
Fluid Membranes
From Alberts et al
Lipid Rafts
From Dykstra et al
Phase 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 peak
Tm: peak position (phase transition temperature)
DT1/2: FWHM of peak
DH: area under the peak (enthalpy of transition)
DS = DH/Tm: entropy of transition
Differential Scanning Calorimetry
• variation of excess specific heat with temperature for
two-state, endothermic process
Differential Scanning Calorimetry
Differential Scanning Calorimetry
Differential Scanning Calorimetry
DSC curves of distearoyl PC
(DSPC) layers as a function
of water content C
Chapman et al., Chem. Phys. Lipids (1967)
Lipid Layer Ordering
Short range order described by
: chains are disordered (melted)
• Trans-gauche isomerization
• Rapid diffusion (translation & rotation)
b: chains stiff, oriented parallel to each other, perpendicular to
bilayer plane
b’: chains tilted with respect to bilayer normal
c: crystalline phase (Lc is lamellar but crystalline within the plane)
Long range order described by
L: 1D lamellar
T: 3D tetragonal
P: 2D rectangular
R: rhombohedral
H: 2D hexagonal
Q: cubic
Lipid Layer Ordering
Lipid Phase Diagram
Phase diagram for PC/water
systems
Blume, Acta ThermChimActa (1991)
Lipid 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
Lipid Phase Transitions
• Can investigate changes in transition temps with chain length, etc.
Blume, Acta ThermChimActa (1991)
Lipid Phase Transitions
Dependence of DH and Tm
on position of double bond
in PCs with chain length of
18 carbons
Nature can control Tm by
placement of double bond
Blume, Acta ThermChimActa (1991)
Influence 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
Lipid Monolayers
Not 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
Langmuir Trough
Schematic of Langmuir trough
Norde, Colloids and Interfaces in Life Sciences (2003)
Surface Pressure-Area Isotherm
G: gas; LE: liquid expanded; LC: liquid condensed; S: solid
Norde, Colloids and Interfaces in Life Sciences (2003)
Phase Coexistence
Brewster angle microscopy of monolayers showing the
Coexistence of LC (light) and LE (dark) phases
Norde, Colloids and Interfaces in Life Sciences (2003)
Compressibility
• 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
Phase Coexistence
Orientations of
amphiphilic molecules
for the various phases
on the pressure-area
isotherms
Norde, Colloids and Interfaces in Life Sciences (2003)
Temperature Dependence of p-A Isotherms
• as temperature increases
• pressure at onset of LE → LC transition increases
• corresponding value of am decreases
• coexistence region decreases
Norde, Colloids and Interfaces in Life Sciences (2003)
Temperature Dependence of p-A Isotherms
p-A isotherms for DPPC at different temperatures
Albrecht et al., J. Phys. (Paris) (1978)
Langmuir-Blodgett Film Formation
• formation of Y-type Langmuir-Blodgett film
• transfer rates of ~1 mm/s
Norde, Colloids and Interfaces in Life Sciences (2003)
Langmuir-Blodgett Film Formation
• X-type transfer
• Z-type transfer
• can also use Langmuir-Schaefer deposition
• horizontal touch of substrate on monolayer
Norde, Colloids and Interfaces in Life Sciences (2003)