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Thorsten Wohland

Publications and source records attributed to Thorsten Wohland.

12 recordsLinked to original sources
PubMed2006

Molecular mechanisms that govern the specificity of Sushi peptides for Gram-negative bacterial membrane lipids.

Factor C-derived Sushi peptides (S1 and S3) have been shown to bind lipopolysaccharide (LPS) and inhibit the growth of Gram-negative bacteria but do not affect mammalian cells. On the premise that the composition of membrane phospholipids differs between the microbial and human cells, we studied the modes of interaction between S1 and S3 and the bacterial membrane phospholipids, POPG, in comparison to that with the mammalian cell membrane phospholipids, POPC and POPE. S1 exhibits specificity against POPG, suggesting its preference for bacterial anionic phospholipids, regardless of whether the phospholipids form vesicles in a solution or a monolayer on a solid surface. The specificity of the Sushi peptides for POPG is a consequence of the electrostatic and hydrophobic forces. The unsaturated nature of POPG confers fluidity to the lipid layer, and being in the proximity of LPS in the microenvironmental milieu, POPG probably enhances the insertion of the peptide-LPS complex into the bacterial inner membrane. Furthermore, during its interaction with POPG, the S1 peptide underwent a transition from random to alpha-helical coil, while S3 became a mixture of beta-sheet and alpha-helical structures. This differential structural change in the peptides could be responsible for their different modes of disruption of POPG vesicles. Conceivably, the selectivity for POPG spares the mammalian membranes from undesirable effects of antimicrobial peptides, which could be helpful in designing and developing a new generation of antibiotics and in offering some clues about the specific function of Factor C, a LPS biosensor.

Anions↗
PubMed2006

Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy.

A fluorescence correlation spectroscopy (FCS) setup is built with an electron multiplying charge-coupled device camera. Although the instrument has a limited time resolution of 4 ms, compared to 0.1-0.2 mus for common instruments using avalanche photodiodes, it allows multiplexing of FCS measurements, has a software-adjustable pinhole after data collection, performs flow speed as well as flow direction measurements in microchannels and could be used to do spectral FCS. Measurements are performed on fluorescent dyes and polystyrene beads in high-viscosity media and on epidermal growth factor receptors in Chinese hamster ovary cells. Using real measurements on single spots, multiplexing of focal spots and detection elements are simulated and the results are discussed.

Animals↗
PubMed2006

Prism-based multicolor fluorescence correlation spectrometer.

We report the design and application of a prism-based detection system for fluorescence (cross) correlation spectroscopy. The system utilizes a single laser wavelength for the simultaneous excitation of several dyes of different emission spectra. Fluorescence light is spectrally separated with a prismatic setup, and wavelengths are selected by scanning a fiber-coupled avalanche photodiode across the image spots. Multicolor autocorrelations are demonstrated with standard and tandem dyes, and fluorescence cross-correlation measurements of biotinylated nanocontainers and streptavidin are presented. This spectrometer offers high optical stability and no focal volume mismatch for the multicolor detection of molecular dynamics and interactions, with single-molecule sensitivity.

Equipment Design↗
PubMed2006

Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect higher order molecular interactions using single wavelength laser excitation.

Fluorescence cross-correlation spectroscopy is a powerful method for the study of molecular interactions and dynamics in solution and even in living cells. Usually, in the optical setup, either two laser beams have to be superimposed in their respective confocal volumes or two-photon excitation is used for a dual-color detection system. It has been shown recently that fluorescence cross correlation can be achieved with spectrally similar fluorophores using single wavelength excitation fluorescence cross-correlation spectroscopy (SW-FCCS). In this study, we show that SW-FCCS allows the simultaneous excitation of up to three fluorophores in which the cross correlation of their fluctuation signals is detected separately in three detection channels. The experimental and theoretical model to describe triple pairwise cross correlations incorporating cross talk and possible changes in emission characteristics such as quenching upon binding are outlined. The effectiveness of SW-FCCS to detect binding of three interacting partners is experimentally verified with a standard ligand-receptor model, biotin-streptavidin, where differently labeled biotin ligands and their binding to a third-color labeled streptavidin are studied. The cross-correlation amplitudes and their changes with stoichiometric binding are analyzed and the upper limits of dissociation constants are determined. Performed with appropriate negative controls, SW-FCCS can determine interaction patterns between ligands and receptors.

Biotin↗
PubMed2006

The molecular mechanism of interaction between sushi peptide and Pseudomonas endotoxin.

Septic shock is caused by Gram-negative bacterial infection. Lipopolysaccharide (LPS) is the bioactive molecule present on the outer membrane of the Gram-negative bacteria. It is generally thought that LPS interacts with sensors on the host cell membrane to activate the intracellular signaling pathway resulting in the overproduction of cytokines such as TNF-alpha. This causes inflammation and ultimately, septic shock. Lipid A is the pharmacophore of the LPS molecule. Thus, developing bio-molecules which are capable of binding LPS at high affinity, especially to the lipid A moiety is an efficient way to neutralize the LPS toxicity. Factor C, a serine protease in the horseshoe crab ameobocytes, is sensitive to trace levels of LPS. We have derived Sushi peptides from the LPS-binding domains of Factor C. Our earlier study showed that the Sushi peptides inhibit LPS-induced septic shock in mice. Here, we demonstrate that the molecular interaction between LPS and Sushi 1 peptide is supported by the hydrophobic interaction between the lipid tail of LPS and Sushi 1 peptide. Furthermore, in the presence of LPS, the peptide transitions from a random structure into an alpha-helical conformation and it disrupts LPS aggregates, hence, neutralizing the LPS toxicity.

Animals↗
PubMed2005

Investigation of a novel artificial antimicrobial peptide by fluorescence correlation spectroscopy: an amphipathic cationic pattern is sufficient for selective binding to bacterial type membranes and antimicrobial activity.

Fluorescence Correlation Spectroscopy (FCS) is used to study the interaction of a recently designed antimicrobial peptide, called V4, with LPS and lipids of varying head and tail groups. V4 is designed based on a known amphipathic cationic pattern BHPHB (B: basic; H: hydrophobic; P: polar residue, respectively) and shows a good combination of high antimicrobial activity, low cytotoxic activity and low hemolytic activity. It is shown that V4 has high binding affinity for LPS, which is the major component of the outer membrane of Gram-negative bacteria, and shows selectivity for negatively charged lipids in contrast to zwitterionic lipids at a low peptide/lipid ratio. At high peptide/lipid ratio, V4 can permeabilize vesicles composed of negatively charged lipids and eventually cause vesicle fusion. The identification of the amphipathic cationic pattern as the mediator of selectivity and antimicrobial activity could be a first step in the rational design of better antimicrobial peptides.

Anti-Infective Agents↗
PubMed2005

Single wavelength excitation fluorescence cross-correlation spectroscopy with spectrally similar fluorophores: resolution for binding studies.

It was shown recently that fluorescence cross-correlation spectroscopy (FCCS) can be performed using a single laser wavelength for excitation (SW-FCCS) [L. C. Hwang and T. Wohland, Chem. Phys. Chem 5, 549 (2004).]. This method simplifies the FCCS setup since it does not require the simultaneous alignment of two lasers to the same focal spot. But up to now the method was shown to work only with dyes possessing large Stokes' shifts, and thus was limited to the use of quantum dots and tandem dyes. In this work we show that standard organic dyes with overlapping emission spectra, for instance fluorescein and tetramethylrhodamine, can be used as fluorescent pairs in SW-FCCS. As a biological model system for ligand-receptor interactions we studied the binding of biotin to streptavidin. To investigate the applicability of SW-FCCS for binding studies we adapt the existing FCCS theory for SW-FCCS and calculate limits for the measurement of dissociation constants in dependence on sample concentration, sample purity, and spectral cross talk between the different detection channels.

Journal Article↗
PubMed2004

Perturbation of Lipopolysaccharide (LPS) Micelles by Sushi 3 (S3) antimicrobial peptide. The importance of an intermolecular disulfide bond in S3 dimer for binding, disruption, and neutralization of LPS.

S3 peptide, derived from the Sushi 3 domain of Factor C, which is the lipopolysaccharide (LPS)-sensitive serine protease of the horseshoe crab coagulation cascade, was shown previously to harbor antimicrobial activity against Gram-negative bacteria. However, the mechanism of action remains poorly understood at the molecular level. Here we demonstrate that the intermolecular disulfide bonding of S3 resulting in S3 dimers is indispensable for its interaction with LPS. The binding properties of the S3 monomer and dimer to LPS were analyzed by several approaches including enzyme-linked immunosorbent assay (ELISA)-based assay, surface plasmon resonance, and fluorescence correlation spectroscopy (FCS). It is evident that the S3 dimer exhibits stronger binding to LPS, demonstrating 50% LPS-neutralizing capability at a concentration of 1 mum. Circular dichroism spectrometry revealed that the S3 peptide undergoes conformational change in the presence of a disulfide bridge, transitioning from a random coil to beta-sheet structure. Using a fluorescence correlation spectroscopy monitoring system, we describe a novel approach for examining the mechanism of peptide interaction with LPS in the native environment. The strategy shows that intermolecular disulfide bonding of S3 into dimers plays a critical role in its propensity to disrupt LPS micelles and consequently neutralize LPS activity. S3 dimers display detergent-like properties in disrupting LPS micelles. Considering intermolecular disulfide bonds as an important parameter in the structure-activity relationship, this insight provides clues for the future design of improved LPS-binding and -neutralizing peptides.

Dimerization↗
PubMed2003

Monitoring expression and clustering of the ionotropic 5HT3 receptor in plasma membranes of live biological cells.

The ionotropic 5HT(3) receptor was expressed in transiently transfected mammalian cells, yielding an unprecedented high concentration of up to 12 million receptors per cell. Receptor traffic in the plasma membrane of live cells was observed continuously over 24 h by fluorescence scanning confocal microscopy. This was possible by using 5HT(3) receptor-specific fluorescent ligands with high binding affinity and low off-rate to pulse label receptors at any time after appearance on the cell surface, and label subsequently those receptors expressed later by another, spectrally distinguishable, high-affinity fluorescent ligand. Having reached a critical cell surface concentration of approximately 3000 receptors/microm(2), the receptors started to aggregate in patches with a 4-fold increased surface concentration. The clusters were constantly delivered from a pool of freshly expressed receptors isotropically distributed within the basolateral region of the cell membrane. From there, they migrated to and accumulated on the apical cell surface approximately 9 h after transfection. Individual clusters grew until they reached a critical size of 1-2 microm when they merged to form with 3-5 microm large macroclusters. Clustered receptors were immobile on the minute time scale but always coexisted with monomeric receptors in the regions surrounding the clusters as revealed by fluorescence correlation spectroscopy. Because the receptor density of 12 000 receptors/microm(2) in the patches is as high as that found in two-dimensional crystals of certain membrane proteins, such patches might be a proper source for direct crystallization of membrane proteins without prior purification.

Cell Line↗
PubMed2002

Functional immobilization of a ligand-activated G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters. They are the largest family of receptors in the human genome and constitute the largest class of targets for drug discovery. To facilitate studies of GPCR activation and interactions with other proteins, we developed a simple method to immobilize a functional, detergent-solubilized GPCR on gold and glass surfaces. The beta(2) adrenergic receptor (beta(2)AR), a prototypical GPCR, was purified and labeled with a reporter fluorophore at a conformationally sensitive site. The detergent-soluble fluorescent beta(2)AR was immobilized through its amino-terminal FLAG epitope on a surface layered with biotinylated bovine serum albumin, avidin, and biotinylated M1 antibody. Agonist activation of the beta(2)AR was monitored in real time by fluorescence microscopy. This approach will make it possible to study conformational dynamics of single immobilized receptors and to generate arrays of functional GPCRs for novel high-throughput screening strategies.

Animals↗
PubMed2002

Analysis of biomolecular interactions using a miniaturized surface plasmon resonance sensor.

A commercially available miniaturized surface plasmon resonance sensor has been investigated for its applicability to biological interaction analysis. The sensor was found to exhibit excellent repeatability and linearity for high-refractive index solutions and good reproducibility for the binding of proteins. Its detection limit for the monoclonal antibody M1 was found to be 2.1 fmol, which corresponds to a surface concentration of 21 pg/mm2. Simple surface immobilization procedures relying on biotin/avidin or glycoprotein/lectin chemistry have been explored. Equilibrium dissociation constants for the binding of the FLAG peptide to its monoclonal antibody (M1) and for the binding of concanavalin A to a glycoprotein have been determined. The close agreement of these measurements with values obtained by surface fluorescence microscopy and fluorescence correlation spectroscopy helps to validate the use of this device. Thus, this sensor shows promise as an inexpensive, portable, and accurate tool for bioanalytical applications in laboratory and clinical settings.

Animals↗