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We study lipid phase behavior using giant unilamellar vesicles to model cell membrane dynamics. In our system, we investigate the effects of cross-linking in the head groups position via biotinylated lipids, avidin, and its analogues. Cross-linking is the linking of two molecules (biotinylated lipids) via a crosslinking agent (avidin). Vesicles allow us to isolate the lipid rearrangement due to cross-linking, a common activity on cell surfaces. By comparing specific binding strength of the coupling and self-adhesion, we study the role that cross-linking plays in membrane behavior. Using anti-avidin we attempt to induce aggregation of the membrane bound protein, producing micron size phase domains from initial one-phase vesicles. Confocal microscopy enables us to image this change in the membrane dynamics. Using phase specific dyes, we probe phase segregation on the nanometer scale from the addition of a cross-linker to the system. Forster Resonance Energy Transfer (FRET) enables us to detect clustering on the submicron (1-10 nm) scale, beyond the limits of conventional microscopy. Both techniques allow us to quantify the phase behavior due presence of the cross-linking agent. Using FRET we detect lipid rearrangement associated with the transition from one-phase vesicles to two-phase vesicles using two different fluorescent dyes, a donor and acceptor. From judicious choice of donor and acceptor dyes, we detect the changes in fluorescence acceptor signal as a function of clustering. We are pursuing lifetime studies to complement our current FRET analyses. From this simple cross-linking system, we model membrane responses to protein complex formation and oligomerization.

We have developed a new time-resolved fluorescence platform which enables us to follow the molecular orientation and dynamics of a lipid monolayer at the air - water interface. Confocal microscopy is limited in its ability to characterize dynamic orientation changes within cellular membranes. By implementing an all reflective Cassegrain objective we minimize dispersion while eliminating the restriction of collinear excitation. This enables us to unambiguously identify fluorescence probe orientation and dynamic freedom within our membrane model, with the highest available temporal resolution, and without the restriction imposed by a supporting substrate. We investigate miscibility transition of ternary lipid mixture, DPPC / DOPC/ Cholesterol, using a combination of fluorescence imaging and time-resolved fluorescence anisotropy. The technique affords unprecedented dynamic characterization for lipid orientation, self-assembly, and dynamic freedom as the monolayer is forced from the liquid to the gel phase. We demonstrate the novelty and applicability of this device by contrasting the time-resolved fluorescence signal of three different lipid probes: 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC), 5-butyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-nonanoic acid (BIODIPY), and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) which show dramatically different orientation and dynamic freedom when bound to the lipid layer, over a range of lipid phases. Using this technique we can resolve highly dynamic processes such as the insertion of peptide and proteins into the lipid membrane.

We study lipid phase behavior using giant unilamellar vesicles to model cell membrane dynamics. In our system, we investigate the effects of cross-linking in the head groups position via biotinylated lipids, avidin, and its analogues. Cross-linking is the linking of two molecules (biotinylated lipids) via a cross-linking agent (avidin). Vesicles allow us to isolate the lipid rearrangement due to cross-linking, a common activity on cell surfaces. By comparing specific binding strength of the coupling and self-adhesion, we study the role that cross-linking plays in membrane behavior. Confocal microscopy gives us the ability to visualize the membrane dynamics. Using phase specific dyes, we study the changes that occur with the addition of a cross-linker to the system. Forster Resonance Energy Transfer (FRET) enables us to detect clustering on the submicron scale, beyond the limits of conventional microscopy. Using FRET we detect lipid rearrangement associated with the transition from one-phase vesicles to two-phase vesicles using two different fluorescent dyes, a donor and acceptor. Both techniques allow us to quantify the phase behavior due presence of the cross-linking agent. From this simple cross-linking system, we model membrane responses to protein complex formation and oligomerization.

We have developed a new time-resolved fluorescence platform which enables us to follow the molecular orientation and dynamics of a lipid monolayer at the air - water interface. This enables us to identify fluorescence probe orientation and dynamic freedom within our membrane model. We have dubbed our technique Dynamic three-Dimensional Fluorescence Microscopy (D3DFM). We demonstrate the novelty and applicability of this device by contrasting the time-resolved fluorescence signal of two different fluorescent probes: NBD-PC and BODIPY bound to a lipid layer (DPPC) and a fatty acid layer (Stearic Acid). We control the phase behavior of our sample by controlling the pressure, and find that unlike the rotational diffusion, the in-plane wobbling is highly sensitive to the the position along the pressure-area isotherm. We believe this is indicative phase coexistence. Other fluorescence techniques are not sensitive to this form of motion due to the geometric constraints of collinear excitation. Using this probe we are able to characterize local dynamic changes that take place upon lipid phase transition, which may be critical for membrane protein recognition and insertion.

The photochemical reaction dynamics of a light-oxygen-voltage (LOV) domain from the blue light sensor protein, FKF1 (flavin-binding Kelch repeat F-box) was studied by means of the pulsed laser-induced transient grating method. The observed absorption spectral changes upon photoexcitation were similar to the spectral changes observed for typical LOV domain proteins (e.g., phototropins). The adduct formation took place with a time constant of 6 μs. After this reaction, a significant conformational change with a time constant of 6 ms was observed as a change in the diffusion coefficient. An FKF1-LOV mutant without the conserved loop connecting helices E and F, which is present only in the FKF1/LOV Kelch protein 2/ZEITLUPE family, did not show these slow phase dynamics. This result indicates that the conformational change in the loop region represents a major change in the FKF1-LOV photoreaction.

In the present work, examples of protein and peptide complexes conformational dynamics, from the solvent state distribution to the gas-phase “de-solvated” state distribution, are characterized for traditionally considered “unstructured” complexes. Conformational motifs and isomerization/conformational dynamics are identified and isomerization kinetics in the ms to few seconds timescale are measured for single molecules using a trapped ion mobility spectrometer - mass spectrometer (TIMS-MS). Theoretical calculations are used to simulate the experimental “TIMS box” single molecule -neutral bath gas phase dynamics and candidate structures are proposed for each conformational state. It is found that, side chain and backbone structural changes are the main motifs governing the conformational inter-conversion processes in the ms-s time scale. Examples will be shown for the case of folded/unfolded protein complexes and DNA-binding proteins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

We investigate peptide interactions with model membranes and how they affect phase dynamics and mobility. Previously, we have studied lipid phase rearrangement due to cross-linking lipids in the headgroup position. Building on this, our current efforts investigate peptide perturbations in lipid bilayers. We cross-link transmembrane peptides on the surface of lipid vesicles to examine the interactions between the helices, mimicking B cell receptor clustering. Expanding on surface-based perturbations, we utilize peptide forming fibrils that associate with anionic lipids, indicating an electrostatic association. These fibril-liposome systems are a useful model to study plaque-based diseases such as Alzheimer's, Parkinson's and type II diabetes. We analyze these associations using microscopy, FRAP, FRET and CD spectroscopy.

The conformational dynamics of intrinsically disordered proteins (IDP's) are inextricably linked to their roles in signaling, regulation, folding, and diseases. Single-molecule methods can contribute valuable information on the conformational dynamics of biomolecules because they allow the observation of unsynchronized dynamics and characterization of diverse populations. Typically, target biomolecules are immobilized to allow study over a longer time window. However, biomolecules with more fluid structures, like IDP's, are highly susceptible to having their structure dominated by the immobilization environment. A method of studying single solution-phase biomolecules for prolonged periods of time would be highly useful for elucidating protein dynamics over many timescales.In this study, we present the use of a microfluidic trap that is capable of canceling Brownian motion to allow the observation of solution-phase dynamics of IDP's over multiple seconds. We will focus on Tau, a protein contributor to the etiology of Alzheimer's disease. Solution-phase conformations of the monomer and small aggregates will be described. The details of the technique, dynamics of the biomolecule targets, and future applications and directions will be discussed.

We investigate peptide interactions with model membranes and how they affect phase dynamics and mobility. Previously, we have studied lipid phase rearrangement due to cross-linking lipids in the headgroup position. Clustering and possible subanomalous diffusion inhibit domain coalescence and alter the conformation energy minimum. Building on this, our current efforts investigate peptide perturbations in lipid bilayers. We cross-link transmembrane peptides on the surface of lipid vesicles to examine the interactions between the helices, mimicking B cell receptor clustering. We conduct these studies by modifying the transmembrane portion of the mIgM receptor and incorporating the resulting peptide into the bilayer. We analyze these associations using microscopy, FRAP, FRET and CD spectroscopy.

We investigate the effects of perturbations on lipid phase dynamics. Our primary tools are confocal microscopy and differential scanning calorimetry (DSC). In coexisting fluid two-phase vesicles we have characterized cross-linking in the fluid disordered phase. Instead of reaching thermodynamic equilibrium, we have documented an increase in meta-stable configurations. Using fluorescence recovery after photobleaching (FRAP), we investigate how cross-linking affects diffusion in lipid bilayer. The diffusion perturbations reveal that cross-linking and non-specific binding slows lateral mobility, which alters lipid dynamics. Since cell membranes are not at thermodynamic equilibrium, our investigations into the dynamics behavior are pertinent to understanding membrane response to common events, such as receptor-ligand complexing, glycosylation, and receptor platform formation.View Large Image | View Hi-Res Image | Download PowerPoint Slide