Biomedical subjects

U Haupts

Publications and source records attributed to U Haupts.

7 recordsLinked to original sources

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

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↗