Biomedical subjects

Wim Crielaard

Publications and source records attributed to Wim Crielaard.

10 recordsLinked to original sources

PubMed2005

Predicting the signaling state of photoactive yellow protein.

As a bacterial blue light sensor the photoactive yellow protein (PYP) undergoes conformational changes upon signal transduction. The absorption of a photon triggers a series of events that are initially localized around the protein chromophore, extends to encompass the whole protein within microseconds, and leads to the formation of the transient pB signaling state. We study the formation of this signaling state pB by molecular simulation and predict its solution structure. Conventional straightforward molecular dynamics is not able to address this formation process due to the long (microsecond) timescales involved, which are (partially) caused by the presence of free energy barriers between the metastable states. To overcome these barriers, we employed the parallel tempering (or replica exchange) method, thus enabling us to predict qualitatively the formation of the PYP signaling state pB. In contrast to the receptor state pG of PYP, the characteristics of this predicted pB structure include a wide open chromophore-binding pocket, with the chromophore and Glu(46) fully solvent-exposed. In addition, loss of alpha-helical structure occurs, caused by the opening motion of the chromophore-binding pocket and the disruptive interaction of the negatively charged Glu(46) with the backbone atoms in the hydrophobic core of the N-terminal cap. Recent NMR experiments agree very well with these predictions.

Bacterial Proteins↗

PubMed2005

Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release.

This study is aimed at the development and application of a convenient and rapid optical assay to monitor the wet-heat resistance of bacterial endospores occurring in food samples. We tested the feasibility of measuring the release of the abundant spore component dipicolinic acid (DPA) as a probe for heat inactivation. Spores were isolated from the laboratory type strain Bacillus subtilis 168 and from two food product isolates, Bacillus subtilis A163 and Bacillus sporothermodurans IC4. Spores from the lab strain appeared much less heat resistant than those from the two food product isolates. The decimal reduction times (D values) for spores from strains 168, A163, and IC4 recovered on Trypticase soy agar were 1.4, 0.7, and 0.3 min at 105 degrees C, 120 degrees C, and 131 degrees C, respectively. The estimated Z values were 6.3 degrees C, 6.1 degrees C, and 9.7 degrees C, respectively. The extent of DPA release from the three spore crops was monitored as a function of incubation time and temperature. DPA concentrations were determined by measuring the emission at 545 nm of the fluorescent terbium-DPA complex in a microtiter plate fluorometer. We defined spore heat resistance as the critical DPA release temperature (Tc), the temperature at which half the DPA content has been released within a fixed incubation time. We found Tc values for spores from Bacillus strains 168, A163, and IC4 of 108 degrees C, 121 degrees C, and 131 degrees C, respectively. On the basis of these observations, we developed a quantitative model that describes the time and temperature dependence of the experimentally determined extent of DPA release and spore inactivation. The model predicts a DPA release rate profile for each inactivated spore. In addition, it uncovers remarkable differences in the values for the temperature dependence parameters for the rate of spore inactivation, DPA release duration, and DPA release delay.

Bacillus↗

PubMed2003

PAS domains. Common structure and common flexibility.

PAS (PER-ARNT-SIM) domains are a family of sensor protein domains involved in signal transduction in a wide range of organisms. Recent structural studies have revealed that these domains contain a structurally conserved alpha/beta-fold, whereas almost no conservation is observed at the amino acid sequence level. The photoactive yellow protein, a bacterial light sensor, has been proposed as the PAS structural prototype yet contains an N-terminal helix-turn-helix motif not found in other PAS domains. Here we describe the atomic resolution structure of a photoactive yellow protein deletion mutant lacking this motif, revealing that the PAS domain is indeed able to fold independently and is not affected by the removal of these residues. Computer simulations of currently known PAS domain structures reveal that these domains are not only structurally conserved but are also similar in their conformational flexibilities. The observed motions point to a possible common mechanism for communicating ligand binding/activation to downstream transducer proteins.

Amino Acid Motifs↗

PubMed2003

Characterization of photocycle intermediates in crystalline photoactive yellow protein.

The photocycle in photoactive yellow protein (PYP) crystals was studied by single-crystal absorption spectroscopy with experimental setups for low-temperature and time-resolved measurements. Thin and flat PYP crystals, suitable for light absorption studies, were obtained using special crystallization conditions. Illumination of PYP crystals at 100 K led to the formation of a photostationary state, which includes at least one hypsochromic and one bathochromic photoproduct that resemble PYP(H) and PYP(B), respectively. The effect of temperature, light color and light pulse duration on the occupancy of these low-temperature photoproducts was determined and appeared similar to that observed in solution. At room temperature a blueshifted photocycle intermediate was identified that corresponds to the blueshifted state of PYP (pB). Kinetic studies show that the decay of this blueshifted intermediate is biphasic at -12 degrees C and 15-fold faster than that observed in solution at room temperature. These altered pB decay kinetics confirm a model that holds that the photocycle in crystals takes place in a shortcut version. In this version the key structural events of the photocycle, such as photoisomerization and reversible protonation of the chromophore, take place, but large conformational changes in the surrounding protein are limited by constraints imposed by the crystal lattice.

Bacterial Proteins↗

PubMed2002

The two photocycles of photoactive yellow protein from Rhodobacter sphaeroides.

The absorption spectrum of the photoactive yellow protein from Rhodobacter sphaeroides (R-PYP) shows two maxima, absorbing at 360 nm (R-PYP(360)) and 446 nm (R-PYP(446)), respectively. Both forms are photoactive and part of a temperature- and pH-dependent equilibrium (Haker, A., Hendriks, J., Gensch, T., Hellingwerf, K. J., and Crielaard, W. (2000) FEBS Lett. 486, 52-56). At 20 degrees C, for PYP characteristic, the 446-nm absorbance band displays a photocycle, in which the depletion of the 446-nm ground state absorption occurs in at least three phases, with time constants of <30 ns, 0.5 micros, and 17 micros. Intermediates with both blue- and red-shifted absorption maxima are transiently formed, before a blue-shifted intermediate (pB(360), lambda(max) = 360 nm) is established. The photocycle is completed with a monophasic recovery of the ground state with a time constant of 2.5 ms. At 7 degrees C these photocycle transitions are slowed down 2- to 3-fold. Upon excitation of R-PYP(360) with a UV-flash (330 +/- 50 nm) a species with a difference absorption maximum at approximately 435 nm is observed that returns to R-PYP(360) on a minute time scale. Recovery can be accelerated by a blue light flash (450 nm). R-PYP(360) and R-PYP(446) differ in their overall protein conformation, as well as in the isomerization and protonation state of the chromophore, as determined with the fluorescent polarity probe Nile Red and Fourier Transform Infrared spectroscopy, respectively.

Bacterial Proteins↗

PubMed2002

Structure of the photoactive yellow protein reconstituted with caffeic acid at 1.16 A resolution.

A structural study is described of the photoactive yellow protein (PYP) reconstituted with the chromophore derivative 3,4-dihydroxycinnamic acid. The crystal structure of PYP reconstituted with this chromophore at 1.16 A resolution is reported in space group P6(5). This is the first high-resolution structure of a photoreceptor containing a modified chromophore. The introduction of an extra hydroxyl group in the native chromophore (i.e. p-coumaric acid) appears to perturb the structure of the hybrid yellow protein only slightly. The chromophore is bound by the protein in two different conformations, separated by a rotation of 180 degrees of the catechol ring. In combination with available spectroscopic data, it is concluded that the caffeic acid chromophore binds to the protein in a strained conformation, which leads to a faster ejection from the chromophore-binding pocket upon pB formation.

Bacterial Proteins↗

PubMed2002

Simplicity in complexity: the photosynthetic reaction center performs as a simple 0.2 V battery.

The photosynthetic reaction center is one of the most complicated molecular complexes. Transducing photon energy to a transmembrane electrochemical potential difference for protons, it is the direct or indirect energy source for virtually all life. We show here that it operates in a simple, battery-like manner, with a maximum potential of 0.20 V. Intriguingly this is only one fifth of the energy of the absorbed photon.

Electric Stimulation↗

PubMed2002

Investigation of in vivo cross-talk between key two-component systems of Escherichia coli.

Intracellular signal transfer in bacteria is dominated by phosphoryl transfer between conserved transmitter and receiver domains in regulatory proteins of so-called two-component systems. Escherichia coli contains 30 such systems, which allow it to modulate gene expression, enzyme activity and the direction of flagellar rotation. The authors have investigated whether, and to what extent, these separate systems form (an) interacting network(s) in vivo, focussing on interactions between four major systems, involved in the responses to the availability of phosphorylated sugars (Uhp), phosphate (Pho), nitrogen (Ntr) and oxygen (Arc). Significant cross-talk was not detectable in wild-type cells. Decreasing expression levels of succinate dehydrogenase (reporting Arc activation), upon activation of the Pho system, appeared to be independent of signalling through PhoR. Cross-talk towards NtrC did occur, however, in a ntrB deletion strain, upon joint activation of Pho, Ntr and Uhp. UhpT expression was demonstrated when cells were grown on pyruvate, through non-cognate phosphorylation of UhpA by acetyl phosphate.

Bacterial Proteins↗

PubMed2002

Cooperativity in signal transfer through the Uhp system of Escherichia coli.

The UhpABC regulatory system in enterobacteria controls the expression of the hexose phosphate transporter UhpT. Signaling is initiated through sensing of extracellular glucose 6-phosphate by membrane-bound UhpC, which in turn modulates the histidine-protein kinase UhpB. Together with the cytoplasmic response regulator UhpA, they constitute a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Activated (i.e., phosphorylated) UhpA binds to the promoter region of uhpT, resulting in initiation of transcription. We have investigated the contribution of transmembrane signaling (through UhpBC) and intracellular activation (through UhpA) to the overall Uhp response (UhpT expression) in vivo. UhpA activation could be made independent of transmembrane signaling when (Delta)uhpBC cells were grown on pyruvate. Inorganic phosphate interfered with glucose 6-phosphate-dependent, UhpBC-mediated, as well as pyruvate-mediated activation of UhpA. The relationship between the concentration of inducer (glucose 6-phosphate) and the Uhp induction rate was nonhyperbolic, indicating positive cooperativity. The degree of cooperativity was affected by the carbon or energy source available to the cells for growth. As pyruvate-mediated activation of UhpA in (Delta)uhpBC cells could result in considerably stronger UhpT expression than glucose 6-phosphate-dependent activation through UhpBC, the observed positive cooperativity for the overall pathway in wild-type cells may reflect the previously described cooperative binding of UhpA to the uhpT promoter (J. L. Dahl et al., J. Biol. Chem. 272:1910-1919, 1997).

Bacterial Proteins↗

PubMed2001

Engineering photocycle dynamics. Crystal structures and kinetics of three photoactive yellow protein hinge-bending mutants.

Crystallographic and spectroscopic analyses of three hinge-bending mutants of the photoactive yellow protein are described. Previous studies have identified Gly(47) and Gly(51) as possible hinge points in the structure of the protein, allowing backbone segments around the chromophore to undergo large concerted motions. We have designed, crystallized, and solved the structures of three mutants: G47S, G51S, and G47S/G51S. The protein dynamics of these mutants are significantly affected. Transitions in the photocycle, measured with laser induced transient absorption spectroscopy, show rates up to 6-fold different from the wild type protein and show an additive effect in the double mutant. Compared with the native structure, no significant conformational differences were observed in the structures of the mutant proteins. We conclude that the structural and dynamic integrity of the region around these mutations is of crucial importance to the photocycle and suggest that the hinge-bending properties of Gly(51) may also play a role in PAS domain proteins where it is one of the few conserved residues.

Amino Acid Sequence↗