Biomedical subjects

Jörg Fitter

Publications and source records attributed to Jörg Fitter.

5 recordsLinked to original sources

PubMed2005

Effects of solubilization on the structure and function of the sensory rhodopsin II/transducer complex.

Lipid-protein interactions are known to play a crucial role in structure and physiological activity of integral membrane proteins. However, current technology for membrane protein purification necessitates extraction from the membrane into detergent micelles. Also, due to experimental protocols, most of the data available for membrane proteins is obtained using detergent-solubilized samples. Stable solubilization of membrane proteins is therefore an important issue in biotechnology as well as in biochemistry and structural biology. An understanding of solubilization effects on structural and functional properties of specific proteins is of utmost relevance for the evaluation and interpretation of experimental results. In this study, a comparison of structural and kinetic data obtained for the archaebacterial photoreceptor/transducer complex from Natronomonas pharaonis (NpSRII/NpHtrII) in detergent-solubilized and lipid-reconstituted states is presented. Laser flash photolysis, fluorescence spectroscopy, and electron paramagnetic resonance spectroscopy data reveal considerable influence of solubilization on the photocycle kinetics of the receptor protein and on the structure of the transducer protein. Especially the protein-membrane proximal region and the protein-protein interfacial domains are sensitive towards non-native conditions. These data demonstrate that relevance of biochemical and structural information obtained from solubilized membrane proteins or membrane protein complexes has to be evaluated carefully.

Archaeal Proteins↗

PubMed2005

Thermostability of irreversible unfolding alpha-amylases analyzed by unfolding kinetics.

For most multidomain proteins the thermal unfolding transitions are accompanied by an irreversible step, often related to aggregation at elevated temperatures. As a consequence the analysis of thermostabilities in terms of equilibrium thermodynamics is not applicable, at least not if the irreversible process is fast with respect the structural unfolding transition. In a comparative study we investigated aggregation effects and unfolding kinetics for five homologous alpha-amylases, all from mesophilic sources but with rather different thermostabilities. The results indicate that for all enzymes the irreversible process is fast and the precedent unfolding transition is the rate-limiting step. In this case the kinetic barrier toward unfolding, as measured by unfolding rates as function of temperature, is the key feature in thermostability. The investigated enzymes exhibit activation energies (E(a)) between 208 and 364 kJmol(-1) and pronounced differences in the corresponding unfolding rates. The most thermostable alpha-amylase from Bacillus licheniformis (apparent transition temperature, T(1/2) approximately 100 degrees C) shows an unfolding rate which is four orders of magnitude smaller as compared with the alpha-amylase from pig pancreas (T(1/2) approximately 65 degrees C). Even with respect to two other alpha-amylases from Bacillus species (T(1/2) approximately 86 degrees C) the difference in unfolding rates is still two orders of magnitude.

Animals↗

PubMed2005

Statistical analysis of diffusion coefficient determination by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) has become an important and widely used technique for many applications in physics, chemistry, and biology. The parameter most frequently addressed by FCS is the diffusion of molecules in solution. Due to the highly non-linear connection between the diffusion coefficient and a measured autocorrelation function, it is extremely difficult to analyse the accuracy of the diffusion-coefficient determination in a FCS experiment. Here, we present a simplified analysis based on some general maximum-likelihood considerations, and numerical result are given for the dependence of the accuracy of the diffusion-coefficient determination on sample concentration, brightness, and measurement time. Optimal concentration values for performing FCS are found.

Journal Article↗

PubMed2004

Art and artefacts of fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is an important technique for studying low concentrations of analyte molecules in solution. The core molecular characteristic that can be addressed by FCS is the translational diffusion coefficient of the analyte molecules, which can be used for i.e. studying molecular binding and reactions, or conformational changes of macromolecules. The present paper discusses several possible optical and photophysical effects that can influence the outcome of a FCS measurement and thus can bias the value of the derived diffusion coefficient.

Artifacts↗

PubMed2003

A measure of conformational entropy change during thermal protein unfolding using neutron spectroscopy.

Thermal unfolding of proteins at high temperatures is caused by a strong increase of the entropy change which lowers Gibbs free energy change of the unfolding transition (DeltaG(unf) = DeltaH - TDeltaS). The main contributions to entropy are the conformational entropy of the polypeptide chain itself and ordering of water molecules around hydrophobic side chains of the protein. To elucidate the role of conformational entropy upon thermal unfolding in more detail, conformational dynamics in the time regime of picoseconds was investigated with neutron spectroscopy. Confined internal structural fluctuations were analyzed for alpha-amylase in the folded and the unfolded state as a function of temperature. A strong difference in structural fluctuations between the folded and the unfolded state was observed at 30 degrees C, which increased even more with rising temperatures. A simple analytical model was used to quantify the differences of the conformational space explored by the observed protein dynamics for the folded and unfolded state. Conformational entropy changes, calculated on the basis of the applied model, show a significant increase upon heating. In contrast to indirect estimates, which proposed a temperature independent conformational entropy change, the measurements presented here, demonstrated that the conformational entropy change increases with rising temperature and therefore contributes to thermal unfolding.

Bacillus↗