Biomedical subjects

C Zscherp

Publications and source records attributed to C Zscherp.

5 recordsLinked to original sources

PubMed2001

Time-resolved FT-IR spectroscopic investigation of the pH-dependent proton transfer reactions in the E194Q mutant of bacteriorhodopsin.

The photoreaction of the E194Q mutant of bacteriorhodopsin has been investigated at various pH values by time-resolved step-scan Fourier-transform infrared difference spectroscopy employing the attenuated total reflection technique. The difference spectrum at pH 8.4 is comparable to the N-BR difference spectra of the wild type with the remarkable exception that D85 is deprotonated. Since the retinal configuration is not perturbed by the E194Q mutation, it is concluded that there is no interaction of D85 with retinal during the lifetime of the N state. At pH 6, a consecutive state to the O intermediate is detected in which D212 is transiently protonated. The comparison with wild-type bacteriorhodopsin reveals that protonation of D212 represents an intermediate step during proton transfer from D85 to the proton release group in the final stage of the reaction cycle. The described effects are more pronounced in the E194Q mutant than in the E204Q mutant demonstrating different roles of these two glutamates/glutamic acids at least in the final stages of the catalytic cycle of bacteriorhodopsin.

Acids↗

PubMed2001

Reaction-induced infrared difference spectroscopy for the study of protein reaction mechanisms.

This paper reviews state-of-the-art reaction-induced infrared difference spectroscopy of proteins. This technique enables detailed characterization of enzyme function on the level of single bonds of proteins, cofactors, or substrates. The following methods to initiate a reaction in the infrared sample are discussed: (i) light-induced difference spectroscopy, (ii) attenuated total reflection with buffer exchange, (iii) the infrared variant of stopped and continuous flow, (iv) temperature and pressure jump, (v) photolytical release of effector substances from caged compounds, (vi) equilibrium electrochemistry, and (vii) photoreduction. Illustrating applications are given including hot topics from the fields of bioenergetics, protein folding, and molecule--protein interaction.

Bacterial Proteins↗

PubMed2000

Substrate binding and enzyme function investigated by infrared spectroscopy.

Protein conformational changes triggered by molecule binding are increasingly investigated by infrared spectroscopy often using caged compounds. Several examples of molecule-protein recognition studies are given, which focus on nucleotide binding to proteins. The investigation of enzyme mechanisms is illustrated in detail using the Ca(2+)-ATPase of the sarcoplasmic reticulum membrane as an example. It is shown that infrared spectroscopy provides valuable information on general aspects of enzyme function as well as on molecular details of molecule-protein interactions and the mechanism of catalysis.

Calcium-Transporting ATPases↗

PubMed1999

In situ determination of transient pKa changes of internal amino acids of bacteriorhodopsin by using time-resolved attenuated total reflection Fourier-transform infrared spectroscopy.

Active proton transfer through membrane proteins is accomplished by shifts in the acidity of internal amino acids, prosthetic groups, and water molecules. The recently introduced step-scan attenuated total reflection Fourier-transform infrared (ATR/FT-IR) spectroscopy was employed to determine transient pKa changes of single amino acid side chains of the proton pump bacteriorhodopsin. The high pKa of D96 (>12 in the ground state) drops to 7.1 +/- 0.2 (in 1 M KCl) during the lifetime of the N intermediate, quantitating the role of D96 as the internal proton donor of the retinal Schiff base. We conclude from experiments on the pH dependence of the proton release reaction and on point mutants where each of the glutamates on the extracellular surface has been exchanged that besides D85 no other carboxylic group changes its protonation state during proton release. However, E194 and E204 interact with D85, the primary proton acceptor of the Schiff base proton. The C==O stretching vibration of D85 undergoes a characteristic pH-dependent shift in frequency during the M state of wild-type bacteriorhodopsin with a pKa of 5.2 (+/-0.3) which is abolished in the single-site mutants E194Q and E204Q and the quadruple mutant E9Q/E74Q/E194Q/E204Q. The double mutation E9Q/E74Q does not affect the lifetime of the intermediates, ruling out any participation of these residues in the proton transfer chain of bacteriorhodopsin. This study demonstrates that transient changes in acidity of single amino acid residues can be quantified in situ with infrared spectroscopy.

Amino Acids↗

PubMedDate not supplied

Investigation of the thermal stability of porin from Paracoccus denitrificans by site-directed mutagenesis and Fourier transform infrared spectroscopy.

The folding of membrane proteins was addressed using outer membrane protein porin from the soil bacterium Paracoccus denitrificans (P. den.). IR spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were used to probe the effect of mutagenesis on the thermal stability of the protein. Secondary structure analysis by amide I ir spectroscopy showed that the wild-type protein was predominantly composed of beta-sheet, which supports the x-ray crystal structure information (A. Hirsch, J. Breed, K. Saxena, O.-M. H. Richter, B. Ludwig, K. Diederichs, and W. Welte, FEBS Letters, 1997, Vol. 404, pp. 208-210). The mutants E81Q, W74C, and E81Q/D148N were shown to have similar secondary structure composition as the wild type. Wild-type protein and the mutants in detergent micelles underwent irreversible denaturation as a result of heating. Transition temperature calculated from the amide I analysis revealed that mutant porins were slightly less stable compared to the wild type. The protein in micelles showed complete monomerization of the trimer above 85 degrees C. In native-like conditions (provided by liposomes), no change was observed in the secondary structure of the protein until 95 degrees C. This is supported by SDS-PAGE as no change in quaternary structure was observed, proving that the proteins are structurally thermostable in liposomes as compared to micelles. Our studies demonstrated that porins resistant to detergents and proteases are highly thermostable as well.

Cell Membrane↗