Biomedical subjects

Marius Schmidt

Publications and source records attributed to Marius Schmidt.

10 recordsLinked to original sources

PubMed2007

Structural basis for the photochemistry of alpha-phycoerythrocyanin.

Phycobiliproteins and phytochromes are light-harvesting and light-sensing proteins containing linear tetrapyrroles, so-called bile chromophores. The chromophores in certain biliproteins, including the phytochromes, isomerize reversibly from a stable Z-configuration to a stable E-configuration when irradiated with light of the appropriate wavelength. Here, we report the crystal structure of alpha-phycoerythrocyanin with its chromophore in the E-configuration, compare it with the Z-configuration found in trimeric phycoerythrocyanin, and reveal the structural bases of the isomerization. The geometric changes of the chromophore account for the large spectral shift, which characterizes the overall transition. Interactions of the chromophore A and D pyrrole rings with flexible protein moieties are required for the formation and stabilization of the isomers. We predict that the results will hold for all photoactive biliproteins.

Crystallography, X-Ray↗

PubMed2005

Local protein flexibility as a prerequisite for reversible chromophore isomerization in alpha-phycoerythrocyanin.

Phycoerythrocyanin is the only cyanobacterial phycobiliprotein containing phycoviolobilin as a chromophore. The phycoviolobilin chromophore is photo-reactive; upon irradiation, the chromophore undergoes a Z/E-isomerization involving the rotation of pyrrole-ring D. We have determined the structure of trimeric phycoerythrocyanin at three different experimental settings: monochromatically at 110 K and 295 K as well as with the Laue method at 288 K. Based on their chemical structures, the restraints for the phycoviolobilin of the alpha-subunit and for the phycocyanobilin chromophores of the beta-subunit were newly generated, which allows a chemically meaningful refinement of both chromophores. All three phycoerythrocyanin structures are very similar; the subunits match within 0.5 A. The detailed comparison of the data obtained with the different measurements provided information about the protein properties around the phycoviolobilin chromophore. For the first time, crystals of a phycobilisome protein are used successfully with the Laue technique. This paves the way for time-resolved macromolecular crystallography, which is able to elucidate the exact mechanisms of the phycoviolobilin photoactivity including the protein involvement.

Crystallography, X-Ray↗

PubMed2005

Ligand migration pathway and protein dynamics in myoglobin: a time-resolved crystallographic study on L29W MbCO.

By using time-resolved x-ray crystallography at room temperature, structural relaxations and ligand migration were examined in myoglobin (Mb) mutant L29W from nanoseconds to seconds after photodissociation of carbon monoxide (CO) from the heme iron by nanosecond laser pulses. The data were analyzed in terms of transient kinetics by fitting trial functions to integrated difference electron density values obtained from select structural moieties, thus allowing a quantitative description of the processes involved. The observed relaxations are linked to other investigations on protein dynamics. At the earliest times, the heme has already completely relaxed into its domed deoxy structure, and there is no photo-dissociated CO visible at the primary docking site. Initial relaxations of larger globin moieties are completed within several hundred nanoseconds. They influence the concomitant migration of photo-dissociated CO to the Xe1 site, where it appears at approximately 300 ns and leaves again at approximately 1.5 ms. The extremely long residence time in Xe1 as compared with wild-type MbCO implies that, in the latter protein, the CO exits the protein from Xe1 predominantly via the distal pocket. A well-defined deligated state is populated between approximately 2 micros and approximately 1 ms; its structure is very similar to the equilibrium deoxy structure. Between 1.5 and 20 ms, no CO is visible in the protein interior; it is either distributed among many sites within the protein or has escaped to the solvent. Finally, recombination at the heme iron occurs after >20 ms.

Amino Acid Substitution↗

PubMed2005

Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds.

Determining 3D intermediate structures during the biological action of proteins in real time under ambient conditions is essential for understanding how proteins function. Here we use time-resolved Laue crystallography to extract short-lived intermediate structures and thereby unveil signal transduction in the blue light photoreceptor photoactive yellow protein (PYP) from Halorhodospira halophila. By analyzing a comprehensive set of Laue data during the PYP photocycle (forty-seven time points from one nanosecond to one second), we track all atoms in PYP during its photocycle and directly observe how absorption of a blue light photon by its p-coumaric acid chromophore triggers a reversible photocycle. We identify a complex chemical mechanism characterized by five distinct structural intermediates. Structural changes at the chromophore in the early, red-shifted intermediates are transduced to the exterior of the protein in the late, blue-shifted intermediates through an initial "volume-conserving" isomerization of the chromophore and the progressive disruption of hydrogen bonds between the chromophore and its surrounding binding pocket. These results yield a comprehensive view of the PYP photocycle when seen in the light of previous biophysical studies on the system.

Bacterial Proteins↗

PubMed2005

Ligand migration and protein fluctuations in myoglobin mutant L29W.

We have determined eight X-ray structures of myoglobin mutant L29W at various experimental conditions. In addition, infrared spectroscopic experiments are presented, which are discussed in the light of the X-ray structures. Two distinct conformations of the CO-ligated protein were identified, giving rise to two stretching bands of heme-bound CO. If L29W MbCO crystals are illuminated around 180 K, a deoxy species is formed. The CO molecules migrate to the proximal side of the heme and remain trapped in the so-called Xe1 cavity upon temperature decrease to 105 K. The structure of this photoproduct is almost identical to the equilibrium high-temperature deoxy Mb structure. If the temperature is cycled to increasingly higher values, CO recombination is observed. Three intermediate structures have been determined during the rebinding process. Efficient recombination occurs only above 180 K, the characteristic temperature for the onset of protein dynamics. Rebinding is remarkably slow because bulky residues His64 and Trp29 block important migration pathways of the CO molecule.

Amino Acid Substitution↗

PubMed2005

A structural pathway for signaling in the E46Q mutant of photoactive yellow protein.

In the bacterial photoreceptor photoactive yellow protein (PYP), absorption of blue light by its chromophore leads to a conformational change in the protein associated with differential signaling activity, as it executes a reversible photocycle. Time-resolved Laue crystallography allows structural snapshots (as short as 150 ps) of high crystallographic resolution (approximately 1.6 A) to be taken of a protein as it functions. Here, we analyze by singular value decomposition a comprehensive time-resolved crystallographic data set of the E46Q mutant of PYP throughout the photocycle spanning 10 ns-100 ms. We identify and refine the structures of five distinct intermediates and provide a plausible chemical kinetic mechanism for their inter conversion. A clear structural progression is visible in these intermediates, in which a signal generated at the chromophore propagates through a distinct structural pathway of conserved residues and results in structural changes near the N terminus, over 20 A distant from the chromophore.

Bacterial Proteins↗

PubMed2005

Protein-ligand interaction probed by time-resolved crystallography.

Time-resolved (TR) crystallography is a unique method for determining the structures of intermediates in biomolecular reactions. The technique reached its mature stage with the development of the powerful third-generation synchrotron X-ray sources, and the advances in data processing and analysis of time-resolved Laue crystallographic data. A time resolution of 100 ps has been achieved and relatively small structural changes can be detected even from only partial reaction initiation. The remaining challenge facing the application of this technique to a broad range of biological systems is to find an efficient and rapid, system-specific method for the reaction initiation in the crystal. Other frontiers for the technique involve the continued improvement in time resolution and further advances in methods for determining intermediate structures and reaction mechanisms. The time-resolved technique, combined with trapping methods and computational approaches, holds the promise for a complete structure-based description of biomolecular reactions.

Bacterial Proteins↗

PubMed2004

Analysis of experimental time-resolved crystallographic data by singular value decomposition.

Singular value decomposition (SVD) separates time-dependent crystallographic data into time-independent and time-dependent components. Procedures for the effective application of SVD to time-resolved macromolecular crystallographic data have yet to be explored systematically. Here, the applicability of SVD to experimental crystallographic data is tested by analyzing 30 time-resolved Laue data sets spanning a time range of nanoseconds to milliseconds through the photocycle of the E46Q mutant of photoactive yellow protein. The data contain random and substantial systematic errors, the latter largely arising from crystal-to-crystal variation. The signal-to-noise ratio of weighted difference electron-density maps is significantly improved by the SVD flattening procedure. Application of SVD to these flattened maps spreads the signal across many of the 30 singular vectors, but a rotation of the vectors partitions the large majority of the signal into only five singular vectors. Fitting the time-dependent vectors to a sum of simple exponentials suggests that a chemical kinetic mechanism can describe the time-dependent structural data. Procedures for the effective SVD analysis of experimental time-resolved crystallographic data have been established and emphasize the necessity for minimizing systematic errors by modification of the data-collection protocol.

Algorithms↗

PubMed2004

Protein kinetics: structures of intermediates and reaction mechanism from time-resolved x-ray data.

We determine the number of authentic reaction intermediates in the later stages of the photocycle of photoactive yellow protein at room temperature, their atomic structures, and a consistent set of chemical kinetic mechanisms, by analysis of a set of time-dependent difference electron density maps spanning the time range from 5 micros to 100 ms. The successful fit of exponentials to right singular vectors derived from a singular value decomposition of the difference maps demonstrates that a chemical kinetic mechanism holds and that structurally distinct intermediates exist. We identify two time-independent difference maps, from which we refine the structures of the corresponding intermediates. We thus demonstrate how structures associated with intermediate states can be extracted from the experimental, time-dependent crystallographic data. Stoichiometric and structural constraints allow the exclusion of one kinetic mechanism proposed for the photocycle but retain other plausible candidate kinetic mechanisms.

Bacterial Proteins↗

PubMed2003

Application of singular value decomposition to the analysis of time-resolved macromolecular x-ray data.

Singular value decomposition (SVD) is a technique commonly used in the analysis of spectroscopic data that both acts as a noise filter and reduces the dimensionality of subsequent least-squares fits. To establish the applicability of SVD to crystallographic data, we applied SVD to calculated difference Fourier maps simulating those to be obtained in a time-resolved crystallographic study of photoactive yellow protein. The atomic structures of one dark state and three intermediates were used in qualitatively different kinetic mechanisms to generate time-dependent difference maps at specific time points. Random noise of varying levels in the difference structure factor amplitudes, different extents of reaction initiation, and different numbers of time points were all employed to simulate a range of realistic experimental conditions. Our results show that SVD allows for an unbiased differentiation between signal and noise; a small subset of singular values and vectors represents the signal well, reducing the random noise in the data. Due to this, phase information of the difference structure factors can be obtained. After identifying and fitting a kinetic mechanism, the time-independent structures of the intermediates could be recovered. This demonstrates that SVD will be a powerful tool in the analysis of experimental time-resolved crystallographic data.

Algorithms↗