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Biological Nanomaterials NANO*4100 FALL 2014 Instructor:John Dutcher Office:MacN 451 Phone + phone mail : Ext. 53950 E-mail:dutcher@uoguelph.ca Web:www.physics.uoguelph.ca/psi Lectures:M W F13:30 14:20MacN 201 Course Website: http://www.physics.uoguelph.ca/~dutcher/nano4100/

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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 self- assembly of aggregates of biological molecules and nanomaterials Appreciate the diversity and complexity of self- assembled biological nanomaterials Expand scientific writing skills to develop effective communication

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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

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Evaluation Problem Assignments30% Directed Reading Assignments 15% Marking of NANO*1000 Report 5% Midterm Test20% Final Examination30% ____________________________________ Total100%

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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

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Guest Instructors Rob Wickham (Physics):copolymers Leonid Brown (Physics):proteins Doug Fudge (MCB):protein filaments

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liquid crystals surfactants colloids polymers biopolymers cells foods Soft Materials

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bonding between molecules is weak comparable to thermal energy k B T ~ 1/40 eV (@RT) can have big changes to soft materials with small changes in environment temperature, pH, ionic strength, applied fields

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Soft Materials hydrogels C. Chang et al. Euro Polym J 46, 92 (2010) Swollen in water As-prepared Dried Swollen in NaCl solution

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Soft Materials rubber elasticity T. Russell, Science 297, 964 (2002) Stretched Unstretched

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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)

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Large Range of Length Scales properties depend on length scale of measurement complex, hierarchical structure processing is the key [P. Ball, Made to Measure]

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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

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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

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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

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Quantitative Biology simple harmonic oscillator Phillips et al., Physical Biology of the Cell

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Quantitative Biology different levels of modeling beyond the spherical cow Phillips et al., Physical Biology of the Cell DNA membrane

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Rules of Thumb Phillips et al., Physical Biology of the Cell

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Rules of Thumb Phillips et al., Physical Biology of the Cell

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Drunkards walk Courtesy of George Gamow Random Walks

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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

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N = 1000 (a)Gaussian random walk random walk (b) self-avoiding random walk random walk Polymer Conformation a b

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Self-Similarity of a Polymer Molecule

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Swimming of Bacteria

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Contribution of Physical Science to Biology Is Hard to OverestimateX-ray NMR ESR EM PDE RGS9-1 G t/i1 -1.5+1.5 -5.5+5.5 ppm ( 1 H) ppm ( 13 C) from Ridge et al.

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Case Study of Bacteriorhodopsin - Contribution of Physical Methods from Luecke et al. 7 transmembrane helices light-driven ion pump Youtube video on bacteriorhodopsin from Alberts et al.

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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

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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

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Dynamics of Cells Youtube video on the Inner Life of the Cell from Biovisions project @ Harvard Swimming bacteria (Howard Berg) Pilus retraction (Howard Berg)

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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

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Biological Membranes Cryo-electron microscopy reveals detailed structure Phillips V. Matias, U of Guelph PhD thesis (A) (A) C. crescentus (B) (B)Intestinal epithelial cells (C) (C)Photoreceptors in rod cell (D) (D)Mitochondrian surrounded by endoplasmic reticulum S. aureus septum

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Major Components of a MembraneMembraneProteins LipidBilayer Characteristic molecular weights Lipids: 0.5-2 kDa Proteins: 5-6000 kDa from Luecke et al. Other components: carbohydrates, water, ions

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Fluid Mosaic Model From Cooper Singer & Nicolson, Science (1972)

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Evolution of Membrane Models Phillips, Physical Biology of the Cell Sackmann (1995) Singer & Nicolson (1972) Israelachvili (1978)

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Restrictions to Free Diffusion of Membrane Proteins from Vereb et al. A lipid microdomains B, C cytoskeleton D protein association

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Hydration of a Lipid Bilayer (MD Simulation) from Popot and Engelman

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Membrane Proteins and Lipids Are Often Linked with Carbohydrates (glycoproteins and glycolipids) From Lodish et al

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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)

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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 PC: phosphatidylcholine or lecithin PE: phosphatidylethanolamine PG: phosphatidylglycerol PI: phosphatidylinositol neutralcharged

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Building a Lipid Molecule polarhydrophobic 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

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Building a Lipid Molecule polarhydrophobic DMPC lipid: di-acylglycerol & phosphatidylcholine lysolipid Phosphatic acid phosphate glycerol choline From Mouritsen

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Phospholipids: Structure Overview Typical Phospholipid Amphipathic Nature! Polar, Hydrophilic Non-Polar, Hydrophobic Variable From Renninger

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Major Phospholipids From Alberts et al glycerol phosphate choline

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Major Phospholipids From Mouritsen

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Major Phospholipids From Mouritsen

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Glyco(sphingo)lipids From Alberts et al

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Cholesterol Stiffens Fluid Membranes From Alberts et al

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Lipid Rafts From Dykstra et al

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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 T m : peak position (phase transition temperature) T 1/2 : FWHM of peak H: area under the peak (enthalpy of transition) S = H/T m : entropy of transition

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Differential Scanning Calorimetry variation of excess specific heat with temperature for two-state, endothermic process

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Differential Scanning Calorimetry

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DSC curves of distearoyl PC (DSPC) layers as a function of water content C Chapman et al., Chem. Phys. Lipids (1967)

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Lipid Layer Ordering Short range order described by : chains are disordered (melted) Trans-gauche isomerization Rapid diffusion (translation & rotation) : chains stiff, oriented parallel to each other, perpendicular to bilayer plane : chains tilted with respect to bilayer normal c: crystalline phase (L c is lamellar but crystalline within the plane) Long range order described by L: 1D lamellarT: 3D tetragonal P: 2D rectangularR: rhombohedral H: 2D hexagonalQ: cubic

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Lipid Layer Ordering

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Lipid Phase Diagram Blume, Acta ThermChimActa (1991) Phase diagram for PC/water systems

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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

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Lipid Phase Transitions Can investigate changes in transition temps with chain length, etc. Blume, Acta ThermChimActa (1991)

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Lipid Phase Transitions Blume, Acta ThermChimActa (1991) Dependence of H and T m on position of double bond in PCs with chain length of 18 carbons Nature can control T m by placement of double bond

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Influence of Polar Head Group PEs have a higher T m 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 T m decreases from 63 o C to 41 o C for DPPE PG negatively charged in high ionic strength solvent, charges are shielded at neutral pH, T m, H and S for PGs are similar to those for PCs PS at neutral pH, 2 negative charges and 1 positive charge T m influenced by pH and ionic strength

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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 This allows measurement of the -A isotherm, which has characteristic shape for each type of molecule and provides information about the orientation and packing of the molecules

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Langmuir Trough Norde, Colloids and Interfaces in Life Sciences (2003) Schematic of Langmuir trough

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Surface Pressure-Area Isotherm Norde, Colloids and Interfaces in Life Sciences (2003) G: gas; LE: liquid expanded; LC: liquid condensed; S: solid

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Phase Coexistence Norde, Colloids and Interfaces in Life Sciences (2003) Brewster angle microscopy of monolayers showing the Coexistence of LC (light) and LE (dark) phases

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Compressibility slope of -A isotherm is measure of isothermal compressibility monolayer in gas state is highly compressible but it is less in more condensed states

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Phase Coexistence Norde, Colloids and Interfaces in Life Sciences (2003) Orientations of amphiphilic molecules for the various phases on the pressure-area isotherms

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Temperature Dependence of -A Isotherms Norde, Colloids and Interfaces in Life Sciences (2003) as temperature increases pressure at onset of LE LC transition increases corresponding value of a m decreases coexistence region decreases

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Albrecht et al., J. Phys. (Paris) (1978) -A isotherms for DPPC at different temperatures Temperature Dependence of -A Isotherms

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Langmuir-Blodgett Film Formation Norde, Colloids and Interfaces in Life Sciences (2003) formation of Y-type Langmuir-Blodgett film transfer rates of ~1 mm/s

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Langmuir-Blodgett Film Formation Norde, Colloids and Interfaces in Life Sciences (2003) X-type transfer Z-type transfer can also use Langmuir-Schaefer deposition horizontal touch of substrate on monolayer