Biomedical subjects

U Haupts

Publications and source records attributed to U Haupts.

12 recordsLinked to original sources

PubMed2001

Single-molecule detection technologies in miniaturized high throughput screening: binding assays for g protein-coupled receptors using fluorescence intensity distribution analysis and fluorescence anisotropy.

G Protein-coupled receptors (GPCRs) represent one of the most important target classes for drug discovery. Various assay formats are currently applied to screen large compound libraries for agonists or antagonists. However, the development of nonradioactive, miniaturizable assays that are compatible with the requirements of ultra-high throughput screening (uHTS) has so far been slow. In this report we describe homogeneous fluorescence-based binding assays that are highly amenable to miniaturization. Fluorescence intensity distribution analysis (FIDA) is a single-molecule detection method that is sensitive to brightness changes of individual particles, such as those induced by binding of fluorescent ligands to membrane particles with multiple receptor sites. As a confocal detection technology, FIDA inherently allows reduction of the assay volume to the microliter range and below without any loss of signal. Binding and displacement experiments are demonstrated for various types of GPCRs, such as chemokine, peptide hormone, or small-molecule ligand receptors, demonstrating the broad applicability of this method. The results correlate quantitatively with radioligand binding data. We compare FIDA with fluorescence anisotropy (FA), which is based on changes of molecular rotation rates upon binding of fluorescent ligands to membranes. While FA requires a higher degree of binding, FIDA is sensitive down to lower levels of receptor expression. Both methods are, within these boundary conditions, applicable to uHTS.

Drug Evaluation, Preclinical↗

PubMed1999

Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation.

Multiphoton excitation (MPE) of fluorescent probes has become an attractive alternative in biological applications of laser scanning microscopy because many problems encountered in spectroscopic measurements of living tissue such as light scattering, autofluorescence, and photodamage can be reduced. The present study investigates the characteristics of two-photon excitation (2PE) in comparison with confocal one-photon excitation (1PE) for intracellular applications of fluorescence correlation spectroscopy (FCS). FCS is an attractive method of measuring molecular concentrations, mobility parameters, chemical kinetics, and fluorescence photophysics. Several FCS applications in mammalian and plant cells are outlined, to illustrate the capabilities of both 1PE and 2PE. Photophysical properties of fluorophores required for quantitative FCS in tissues are analyzed. Measurements in live cells and on cell membranes are feasible with reasonable signal-to-noise ratios, even with fluorophore concentrations as low as the single-molecule level in the sampling volume. Molecular mobilities can be measured over a wide range of characteristic time constants from approximately 10(-3) to 10(3) ms. While both excitation alternatives work well for intracellular FCS in thin preparations, 2PE can substantially improve signal quality in turbid preparations like plant cells and deep cell layers in tissue. At comparable signal levels, 2PE minimizes photobleaching in spatially restrictive cellular compartments, thereby preserving long-term signal acquisition.

Animals↗

PubMed1999

Closing in on bacteriorhodopsin: progress in understanding the molecule.

Bacteriorhodopsin is the best understood ion transport protein and has become a paradigm for membrane proteins in general and transporters in particular. Models up to 2.5 A resolution of bacteriorhodopsin's structure have been published during the last three years and are basic for understanding its function. Thus one focus of this review is to summarize and to compare these models in detail. Another focus is to follow the protein through its catalytic cycle in summarizing more recent developments. We focus on literature published since 1995; a comprehensive series of reviews was published in 1995 (112).

Bacteriorhodopsins↗

PubMed1999

Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy.

Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule detection (SMD) techniques and related approaches, such as fluorescence correlation spectroscopy (FCS), stochastically sample the fluorescence properties of individual constituent molecules and only then average many such detection events to define the properties of the assay system as a whole. Analysis of single molecular events is accomplished using confocal optics with an illumination/detection volume of approximately 1 fl (10(-15) L) such that the signal is insensitive to miniaturization of HTS assays to 1 µl or below. In this report we demonstrate the general applicability of one SMD technique (FCS) to assay configuration for target classes typically encountered in HTS and confirm the equivalence of the rate/equilibrium constants determined by FCS and by macroscopic techniques. Advantages and limitations of the current FCS technology, as applied here, and potential solutions, particularly involving alternative SMD detection techniques, are also discussed.

Journal Article↗

PubMed1998

Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy.

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pKa = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 microseconds at pH 7 to 45 microseconds at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 microseconds and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.

Fluorescence↗

PubMed1997

Fluorescence correlation spectroscopy: diagnostics for sparse molecules.

The robust glow of molecular fluorescence renders even sparse molecules detectable and susceptible to analysis for concentration, mobility, chemistry, and photophysics. Correlation spectroscopy, a statistical-physics-based tool, gleans quantitative information from the spontaneously fluctuating fluorescence signals obtained from small molecular ensembles. This analytical power is available for studying molecules present at minuscule concentrations in liquid solutions (less than one nanomolar), or even on the surfaces of living cells at less than one macromolecule per square micrometer. Indeed, routines are becoming common to detect, locate, and examine individual molecules under favorable conditions.

Biophysics↗

PubMed1997

Chloride and proton transport in bacteriorhodopsin mutant D85T: different modes of ion translocation in a retinal protein.

Replacement of aspartate 85 (D85) in bacteriorhodopsin (BR) by threonine but not be asparagine creates at pH<7 an anion-binding site in the molecular similar to that in chloride pump halorhodopsin. Binding of various anions to BR-D85T causes a blue shift of the absorption maximum by maximally 57 nm. Connected to this color change is a change in the absorption difference spectrum of the initial state and the longest living photo intermediate from a positive difference maximum at 460 nm in the absence of transported anions to one at 630 nm in their presence. Increasing anion concentration cause decreasing decay times of this intermediate. At physiological pH, BR-D85T but not BR-D85N transports chloride ions inward in green light, protons outward in blue or green light and protons inward in white light (directions refer to the intact cell). The proton movements are observable also in BR-D85N. Thus, creation of an anion-binding site in BR is responsible for chloride transport and introduction of anion-dependent spectroscopic properties at physiological pH. The different transport modes are explained with the help of the recently proposed IST model, which states that after light-induced isomerization of the retinal an ion transfer step and an accessibility change of the active site follow. The latter two steps occur independently. In order to complete the cyclic event, the accessibility change, ion transfer and isomerization state have to be reversed. The relative rates of accessibility changes and ion transfer steps define ultimately the vectoriality of ion transfers. All transport modes described here for the same molecule can satisfactorily be described in the framework of this general concept.

Anions↗

PubMed1997

General concept for ion translocation by halobacterial retinal proteins: the isomerization/switch/transfer (IST) model.

Bacteriorhodopsin (BR), which transports protons out of the cell in a light-driven process, is one of the best-studied energy-transducing proteins. However, a consensus on the exact molecular mechanism has not been reached. Matters are complicated by two experimental facts. First, recent results using BR mutants (BR-D85T) and the homologous protein sensory rhodopsin I demonstrate that the vectoriality of active proton transport may be reversed under appropriate conditions. Second, in BR-D85T as well as in the homologous halorhodopsin, protons and chloride ions compete for transport; e.g. the same molecule may transport either a positive or a negative ion. To rationalize these results, we propose a general model for ion translocation by bacterial rhodopsins which is mainly based on two assumptions. First, the isomerization state of the retinylidene moiety governs the accessibility of the Schiff base in the protein; e.g. all-trans, 15-anti, and 13-cis-15-anti direct the Schiff base to extracellular and cytoplasmic accessibility, respectively, but change in accessibility (called the "switch") is a time-dependent process in the millisecond time range. A light-induced change of the isomerization state induces not only a change in accessibility but also an ion transfer reaction. Second, we propose that these two processes are kinetically independent, e.g. that relative rate constants in a given molecule determine which process occurs first, ultimately defining the vectoriality of active transport.

Bacterial Proteins↗

PubMed1996

Different modes of proton translocation by sensory rhodopsin I.

The membrane-bound complex between sensory rhodopsin I (SRI) and its transducer HtrI forms the functional photoreceptor unit that allows transmission of light signals to the flagellar motor. Although being a photosensor, SRI, the mutant SRI-D76N and the HtrI-SRI complex can transport protons, as we demonstrate by using the sensitive and ion-specific black lipid membrane technique. SRI sustains an orange light-driven (one-photon-driven) outward proton transport which is enhanced by additional blue light (two-photon-driven). The vectoriality of the two-photon-driven transport could be reversed at neutral pH from the outward to the inward direction by switching the cut-off wavelength of the long wavelength light from 550 to 630 nm. The cut-off wavelength determining the reversal point decreases with decreasing pH. The currents could be enhanced by azide. A two-photon-driven inward proton transport by SRI-D76N (catalyzed by azide) and by the complex HtrI-SRI is demonstrated. The influence of pH and azide concentration on the rise and decay kinetics of the SRI380 intermediate is analyzed. The different modes of proton translocation of the SRI species are discussed on the basis of a general model of proton translocation of retinal proteins and in the context of signal transduction.

Bacteriorhodopsins↗

PubMed1995

The photoreceptor sensory rhodopsin I as a two-photon-driven proton pump.

Proton translocation experiments with intact cells of Halobacterium salinarium overproducing sensory rhodopsin I (SRI) revealed transport activity of SRI in a two-photon process. The vectoriality of proton translocation depends on pH, being outwardly directed above, and inwardly directed below, pH 5.7. Activation of the transport cycle requires excitation of the initial dark state of SRI, SRI590, to form the intermediate SRI380. Action spectra identify the photocycle intermediates SRI380 and SRI520 as the two photochemically reactive species in the outwardly directed transport process. As shown by flash photolysis experiments, SRI520 undergoes a so-far unknown photochemical reaction to SRI380 with a half-time of <200 micros. Mutation of SRI residue Asp-76, the residue which is equivalent to the proton acceptor Asp-85 in bacteriorhodopsin, to asparagine leads to inactivation of proton translocation. This demonstrates that the underlying mechanisms of proton transport in both retinal proteins share similar features. However, SRI is to our knowledge the first case where photochemical reactions between two thermally unstable photoproducts of a retinal protein constitute a catalytic ion transport cycle.

Journal Article↗

PubMed1995

Chemical reconstitution of a chloride pump inactivated by a single point mutation.

The arginine residue R108 plays an essential role in the transport mechanism of the light-driven anion pump halorhodopsin (HR) as demonstrated by complete inactivation of chloride transport in mutant HR-R108Q. In the presence of substrate anions, guanidinium ions bind to the mutant protein with affinities in the mM range, thereby restoring transport activity and photochemical properties of wild type. One guanidinium ion and one anion are bound per molecule of HR-R108Q. For HR wild type, HR-R108Q-guanidinium and HR-R108K, differences in transport activity and anion selectivity are found which may be explained by effects of anion solvation. The agreement between light-induced FTIR difference spectra of HR wild type and HR-R108Q-guanidinium demonstrates that no structural changes occur in the reconstituted mutant and that the photoreactions of wild type and reconstituted mutant are identical. Furthermore, an IR absorbance band of the guanidino group of R108 can be identified at 1695/1688 cm-1. In HR-R108Q, a guanidinium ion binding close to the mutated residue is proposed to mimick the role of the R108 side chain as the anion uptake site. Thus the wild type reaction mechanism is reconstituted.

Anions↗

PubMed1994

Sensory rhodopsin I photocycle intermediate SRI380 contains 13-cis retinal bound via an unprotonated Schiff base.

Sensory rhodopsin I (SRI), the mutated derivative SRI-D76N and the complex of SRI with its transducer HtrI were overexpressed in Halobacterium salinarium and analyzed by resonance Raman spectroscopy. In the initial state SRI contains all-trans retinal bound via a protonated Schiff base as confirmed by retinal extraction which yields 95 +/- 3% all-trans retinal. The photocycle intermediate absorbing maximally at 380 nm (SRI380) contains a Schiff base linkage between the protein and 13-cis retinal. Extraction of illuminated SRI yields up to 93% 13-cis retinal. Neither the mutation D76N nor HtrI changed the vibrational pattern of the chromophore.

Bacteriorhodopsins↗