Biophysics of Excitable Cells PHYS*2030 FALL 2002

Biological NanomaterialsNANO*4100 FALL 2014Instructor:John DutcherOffice:MacN 451 Phone + phone mail : Ext. 53950E-mail:dutcher@uoguelph.ca 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