1. Langmuir, 2009, DOI:10.1021/la90222g DNA diffusivity decreases with increase in the mole fraction of the gel-phase lipid. Epifluorescence tracking of naked GUVs (top) and microsphere adsorbed GUVs (bottom) reveal binding induced charge separation and slaved motion of lipids. 5µm a b c DMPC DMTAP + Before particle binding After binding Lipids diffusion slaved by particle - Enhanced cooperative binding Particle binding induces DMTAP segregation at liquid phase C DMTAP 15%→50% Isothermal titration calorimetry measures the binding energy quantitatively and shows that binding induces charge separation and facilitates further binding Advance made here: particle binding induces segregation of lipid in membrane and gathers their reins In progress: coupled diffusion of lipids and particles Random walkers on a fluctuating lipid tube For Corrugated Surfaces D~N -1 Model Chain Diffusion on Surfaces For Smooth Surfaces D~N -3/4 Experiments Show D~N -3/2 Include Surface Defects to Explain Data Transition to experimental data when defect spacing is less than chain size DNA mobility on homogeneous bilayers and vesicles Adsorption of ss-DNA on supported cationic lipid bilayer composed of DMTAP or DOTAP Diffusivity of the adsorbed DNA (  ) plotted as a function of the lipid (DMTAP) diffusivity. Also shown is the diffusivity of bacteriorhodopsin in liposomes plotted as a function of lipid mobility in the presence of protein at different Lipid(L)/Protein(P) ratios. L/P = 140 (  ) and L/P = 30 (  ) (adapted from Peters et al,PNAS, 79,4317( 1982)) Mobility of ss DNA tracks lipid mobility Diffusion of large DNA on giant unilamellar vesicles Diffusion of linear DNA Diffusion of circular DNA 0.23  m 2 /s 0.2  m 2 /s No D D=0.44  m 2 /s 48.5kbp linear DNA (3.2*10 7 g/mol) trajectory (black) on DOPC DOTAP (10%) GUV. The three smaller red circles show trajectories from same DNA molecule. Scale of this image is 40  m x 40  m. It is exciting to see that the DNA diffusivity differs from spot to spot, and from time to time, even in the absence of external electric field for electrophoresis. 2D projection of 2.6*10 7 g/mol circular DNA trajectory on giant lipid vesicle surface. From a plot of mean- square displacement against time, the straight line implies a translational diffusion coefficient of D = 0.66  m 2 /sec. X2(m2)X2(m2) Conclusions Lipid mobility controls the diffusivity of short biopolymer adsorbates. Formation of raft-inspired lipid bilayers enhances the effectiveness of polyvalent recognition as well as adsorbed enzyme stability. Domain formation in lipid bilayers and biomolecule recognition can be actively controlled by adding divalent cations. Domains in lipid bilayers provide control over the adsorption and transport of DNA. Nanoparticles stabilize phospholipid vesicles by preventing vesicle fusion even at high vesicle concentrations. Nanoparticles do not interfere with receptor binding or functionalization of bilayer lipids. Nanoparticle binding can locally induce phase transitions in lipid membranes. Education and Outreach: The project has already contributed to the training and development of four graduate students (Krishna Athmakuri, Jeffrey Litt, Chakradhar Padala, and Yan Yu), one undergraduate student (Andrew Devine) and a high school student (Kevin Crimmins). Students are introduced to an interdisciplinary research environment and gain expertise in topics ranging from soft materials to nanotechnology, biophysics, transport phenomena, and biomaterials. Further training is provided through outreach efforts, such as presentations to high school students in the New Visions High School Program and to a high school teacher, Ms. Tammy Borland. Confocal Images of GUVs containing i)5% and ii-iv)20% cholesterol. Characterization by FRET of peptide clustering due to cholesterol dependent phase separation Controlling Biomolecule Stability and Recognition Using Raft-Mimetic Lipid Bilayers Actively Induced Phase Separation Enhanced efficiency of recognition using raft-mimetic liposomes Phase separation leads to a 2 order of magnitude increase in polyvalent recognition Phase separation provides a general mechanism to increase efficiency of polyvalent recognition Confocal image of Ca 2+ induced phase separation of a GUV Peptide-functionalized lipid Gel-Phase lipids Fluid-Phase lipids Angew. Chem. 119, 2257 (2007) No cholesterol 5% cholesterol20% cholesterol Novelty:Actively reconfigurable, nanostructures to control biomolecule adsorption and transport. This biologically-inspired approach seeks to implement, in the bioseparations context, the concept of lipid rafts. Transformative potential: The field of bio-separations is limited by use of passive surfaces. Our goal here is to remove this limitation. To accomplish this task, fundamental underpinnings are under development, those needed to design reconfigurable nanostructured surfaces that enable the separation of biomolecules in a manner that is far more facile and efficient than conventional strategies. Potential Impact on Industry and Society: The understanding of biomolecule recognition and transport provided by this work will impact the design of novel technologies for biosensing, bioseparation, drug delivery, as well as the design of novel therapeutics. The project will also contribute to the training of graduate, undergraduate, and high school students and expose them to a stimulating interdisciplinary research environment. Novel materialsTransportRaft-mimetic bilayers A + + + DNA mobility on heterogeneous bilayers DNA adsorption and diffusion on heterogeneous bilayers Fluorescence micrographs of DNA adsorbed on bilayers consisting of: (a) 10 mole% DSPC and (b) 30 mole% DSPC. (c) Diffusivity of DNA adsorbed on heterogeneous supported bilayers as a function of mole% of the gel-phase lipid DSPC in the bilayer. DNA electrophoresis on heterogeneous bilayers Fluorescence micrographs of 84mer ssDNA labeled with Texas Red dye in the presence of an electric field. (a) t = 0 min, (b) t =8 min and (c) t =16 min DNA diffusivity (D) decreased with size (N) whereas drift velocity is nearly independent of N. Isothermal titration calorimetry shows the affinity of nanoparticles to lipid membrane depends on the surface charge of particles. The binding can be divided into two categories: enthalpy driven, and entropy driven, accordingly. The binding strength is >>kT. Positively charged particles C A Negatively charged particles Enthalpy driven Entropy driven 0 100 200 600 B A Dielectric environment sensitive fluorescence demonstrates the ability of nanoparticles to locally induce liquid-gel phase transition in lipid membranes, which is the driven force for particle binding. Negatively charged particles, Liquid-gel Positively charged particles,Gel->liquid Advance made here: particle binding modulate the membrane structure significantly In progress: particle packing and induced local curvature gel θ - + liquid + - θ - + electrostatic Particle binding induces local phase transition in lipid membranes PNAS,105, 18171-18175 (2008) B D Advance made here: cationic nanoparticles stabilize phospholipid vesicles up to dense volume fractions without fusion, allowing ligand-receptor binding. In progress: interactions with DNA, especially plasmid DNA. Soft Matter 3, 551 (2007) J. Phys. Chem. C 111, 8233 (2007) Fluorescence autocorrelation functions showing that streptavidin binds effectively to vesicle-attached biotin even when nanoparticles stabilize the liposomes to which biotin is attached. 500 nm Δπ=300 mM Δπ=40 0 mM t=0 min t=20 min Δπ=200 mM Δπ=10 0 mM Epifluorescence images of naked GUVs (top) and nanoparticle-stabilized GUVs (bottom) reveal enhanced osmotic stress tolerance for the latter. Nanoparticle-stiffened phospholipid vesicles DiIFITC-SBPMerged Heterogeneous (rapid quench) Heterogeneous (slow quench) Homogeneous Enzyme Adsorption onto Raft-Like Domains Increases Stability JACS 131, 7107 (2009) Adsorption of SBP onto small domains (5 nm) imparts greater stability than larger domains (13 nm) NIRT: Actively Reconfigurable Nanostructured Surfaces for the Improved Separation of Biological Macromolecules Ravi Kane, Steve Granick, and Sanat Kumar, CBET - 0608978 Particle binding induces charge separation and slaved motion