Session D13: Focus Session: Spectroscopy of Biomolecules from Isolated Molecules to Cell Environment III

Imaging of protein partitioning in plasma membranes with coexisting fluid phases

The membrane raft hypothesis postulates the existence of lipid bilayer membrane heterogeneities, or domains, important for cellular membrane functioning, including lateral sorting, signaling and trafficking. The \textit{in vivo }characterization of lipid membrane heterogeneities thus far has been challenging. Lipid membrane rafts have been suggested to be enriched in lipids that confer fluid ordered phase like character to these compositional heterogeneities. Lipid model membranes, on the other hand, allow fluorescence imaging of lipid domain coexistence, but so far micron-size coexisting fluid phases have been demonstrated only in simple, including ternary, lipid mixtures. We found that giant \textit{plasma membrane} \textit{vesicles} (PMVs) obtained from cultured rat basophilic leukemia cells can phase segregate into optically resolvable micron-size phases that are identified as both being fluid. We examined the partitioning of fluorescent lipid analogs and hydrophobic membrane markers and found them to be similar to the partitioning behavior in model membrane systems that show coexisting fluid ordered and fluid disordered phases. Significant temperature dependence of the tendency of PMVs to phase separate was found and analyzed. An advantage of these PMVs is that they contain, at least a large subfraction, of proteins associated with native plasma membranes. We are therefore able to study the partitioning of membrane proteins redistributing between coexisting fluid membrane phases. Trans-membrane proteins, outer leaflet associated peripheral and cytosolic peripheral membrane proteins were examined for their fluid phase partitioning. We suggest our method as a new approach for studying aspects of biological membrane heterogeneity.   

Spectroscopic Characterization of ssDNA Brushes

We use X-ray photoelectron (XPS) and Fourier transform infrared (FTIR) spectroscopies to quantitatively characterize the structure and conformation of single-stranded DNA (ssDNA) brushes on gold surfaces. Self-assembly of these brushes exploits an intrinsic affinity between blocks of adenine nucleotides and gold surfaces. Using brushes of model block oligonucleotides, d(Tm-An), with systematically varied lengths of the thymine and adenine blocks [d(T) and d(A)], we demonstrate that this immobilization strategy enables independent and deterministic control of the grafting density (lateral spacing) and brush conformation. Quantitative analysis of XPS and IR spectroscopic signatures of the model d(Tm-An) brushes confirms that the d(T) blocks extend away from the surface in a brush-like conformation, at lateral spacing 2-3 times larger (grafting density 5-10 times lower) than in analogous films immobilized via standard thiol linkers. The conventional S-Au attachment strategy requires near-saturation grafting densities to adopt this upright conformation, therefore ssDNA immobilization via d(A) blocks offers a unique pathway to achieve a brush-like conformation at low grafting density.   

Detection of $\beta -$Amyloid Peptide Dimer in Solution by Fluorescence Resonance Energy Transfer

Studies have suggested that there is a connection between {\ss}-amyloid-derived diffusible ligands (ADDLs), small oligomers formed from clustering of peptides with 39-42 amino acid units, and pathogenicity of Alzheimer's disease. It is believed that the soluble ADDL oligomers eventually coagulate and precipitate into fibrils that cause neurotoxicity. Although there have been studies characterizing the fibrils structure and the large coagulate formation kinetics, little experimental information exists for the oligomers in the solution phase. We report here the use of fluorescence resonance energy transfer detected through a confocal microscope under single molecule conditions for the detection of the $\beta $-amyloid (1-40) peptide dimer in solution. The structure of the dimer is characterized in terms of the distance of the two N-terminals.   

Nanolaser Spectroscopy of Genetically Engineered Yeast: New Tool for a Better Brew?

A basic function of the cell membrane is to selectively uptake ions or molecules from its environment to concentrate them into the interior. This concentration difference results in an osmostic pressure difference across the membrane. Ultimately, this pressure and its fluctuation from cell to cell will be limited by the availability and fluctuations of the solute concentrations in solution, the extent of inter-cell communication, and the state of respiring intracellular mitochondria that fuel the process. To measure these fluctuations, we have employed a high-speed nanolaser technique that samples the osmotic pressure in individual yeast cells and isolated mitochondria. We analyzed 2 yeast cell strains, normal baker’s yeast and a genetically-altered version, that differ only by the presence of mitochondrial DNA. The absence of mitochondrial DNA results in the complete loss of all the mtDNA-encoded proteins and RNAs, and loss of the pigmented, heme-containing cytochromes. These cells have mitochondria, but the mitochondria lack most normal respiratory chain complexes. The frequency distributions in the nanolaser spectra produced by wild-type and modified cells and mitochondria show a striking shift from Gaussian to Poissonian distributions, revealing a powerful novel method for studying statistical physics of yeast.   

The Influence of Environment on the Reactivity, Dynamics and Spectroscopy of B12 Coenzymes.

Adenosylcobalamin (AdoCbl) dependent enzymes catalyze a variety of chemically difficult reactions that proceed by mechanisms involving organic radicals. In these enzymes radicals are initially generated by homolysis of the cobalt-carbon bond to produce an adenosyl radical and a cob(II)alamin radical. This radical pair may also be generated by optical excitation of the AdoCbl cofactor with visible light. In the work presented here, time-resolved spectroscopic measurements spanning the time range from 10 fs to 10 ns are used to investigate the energetics and dynamics of AdoCbl and other cobalamins as a function of environment. These studies probe the influence of environment on the energy of the low-lying charge transfer states of the cobalamin and on the barriers for dissociation and for recombination of the geminate radical pair. When the AdoCbl coenzyme is bound to the enzyme glutamate mutase, the protein environment is found to stabilize the charge transfer state of AdoCbl relative to observations in water and ethylene glycol. However, the intrinsic rate constant for recombination is only slightly smaller than the rate constant measured in free solution, suggesting the protein does not greatly perturb the ground state stability of the cobalt-carbon bond.   

Single molecule views of Nature's nano-machines

We are interested in the perturbational analysis of biological molecules to better understand their mechanisms. Our readout is the fluorescence signal from individual biomolecules, mainly in the form of single molecule fluorescence resonance energy transfer (FRET). We are pioneering approaches to perturb and control biomolecular conformations using external force (combination of single molecule FRET and optical trap) or other biological motifs (DNA hybridization, G-quadruplex, aptamers,.). In this talk, I will present our latest results on mapping the conformational energy landscape of the Holliday junction through simultaneous fluorescence and force measurements. In addition, a new nanomechanical device called single molecule nano-metronome will be discussed with an outlook toward controlling protein conformations using nucleic acids motifs.   

Time-Resolved Protein Superstructure Disassembly at the Single Particle Level

Many proteins are able to spontaneously self-organize \textit{in vivo }into large, complex structures. These superstructures are often disassembled through the action of key enzymes to fulfill a specific biological role. Yet, the mechanisms of disassembly of such complex structures are poorly understood. We demonstrate the ability to monitor the kinetics of the enzyme assisted disassembly of fluorescently labeled clathrin-coated vesicles in a micro-fluidic flow cell using photon burst and correlation spectroscopy.   

Study of DNA uptake locations in single \textit{E. coli} cells

Artificial gene transfer of bacteria, such as \textit{E. coli}, has become the main stream technique in genetic engineering and molecular cell biology studies. In spite of the great improvements in transformation efficiency, some fundamental questions remained to be answered. For instance, what are the DNA uptake channels and how do they form and function under external stimuli? Furthermore, where are these channels located on the cell membrane? Here we report a study aimed at DNA uptake locations in the two widely used gene transformation techniques: electroporation and heat shock. A direct visualization of the settling location of single DNA molecules inside individual \textit{E. coli} cells was obtained by fluorescence imaging and spectroscopy. Electroporation and heat shock exhibit two distinct characteristics of DNA uptake locations. A preferential distribution toward cell poles during electroporation is consistent with earlier experiments and previously proposed models. However, the result from heat shock is unanticipated in which the majority of DNA enters the cell near the center. Such observation suggests that uptake channels form preferentially where newly-synthesized membrane is located under cation and low temperature treatment   

Real-Time, Nonlinear Optical Probe of Molecular Transport across Living \textit{Escherichia coli} Cell Membranes

We will demonstrate for the first time that a nonlinear optical technique- Second Harmonic Generation- can be used to monitor, with real time resolution, the transport of a molecule across the membranes of a living cell. The transport of the hydrophobic ionic dye molecule malachite green (MG) through both membranes of the gram-negative bacteria \textit{Escherichia coli}, the outer membrane and the cytoplasmic membrane, has been studied. A kinetic model, assuming that the MG molecules penetrate the bacteria outer membrane through classic porin channels while transport across the cytoplasmic membrane is by diffusion through the phospholipid bilayer, is proposed to account for experimental observations. Analysis of the SHG data enables quantitative determination of the transport rate constants and the adsorption equilibrium constants for the \textit{Escherichia coli} cells living in different environments.   

Photon-by-Photon Determination of Emission Bursts from Diffusing Single Chromophores

Diffusing-type single molecule experiment is expected to provide rich information such as protein conformational distribution, DNA sequencing, ultra-sensitive detection, to name a few. However, its application is greatly limited by the difficulty of extracting the useful information out of the noisy data because of the embedded Poissonian noise. Conventional analysis of such trajectories involves further smoothing the data followed by artificially setting a threshold to distinguish the signal, risking the chance of ignoring the fast transition events along the trajectory. Here we report a statistically robust algorithm, which operates on the trajectory photon by photon, based on the well established sequential test model. A demonstration experiment with the gold nanoparticle diffusing throuth the detection volume shows that our algorithm indeed retrieves more information, relieving the incertitude of artificial placement of bin width and threshold.   

Quantum cooperative process in living cells

A model of a quantum cooperative process has accurately accounted for various quantitative observations.$^{1}$ That investigation considered chemical oscillations to be generated by generic quantum oscillators producing discrete quanta with well-defined energy and wavelength. The current work extends the theory by postulating that these oscillations arise from repetitive electron transfers in membranes. We find this produces a limit cycle completely consistent with the hypothetical generic oscillators, accurately reproduces the results of microwave irradiation experiments on yeast, and addresses limits for the smallest possible cell sizes. Questions of coherence in cells and implications for molecular information transfers are briefly considered. $^{1}$R.W. Finkel, \textit{J. Theor. Biol.} in press.