Modeling non-inherited antibiotic resistance.

A mathematical model is presented for the increase and decrease of non-inherited antibiotic resistance levels in bacteria. The model is applied to experimental data on E. coli exposed to amoxicillin or tetracyclin in different concentrations. The parameters of the model are estimated using a Monte Carlo Markov Chain method. The model accurately describes build-up and decline of antibiotic resistance caused by physiological adaptations as long as no genetic changes have occurred. The main conclusion of the analysis is that short time periods are sufficient to re-obtain low MIC-values after long-lasting exposure to these antibiotics.

Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein.

Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira halophila. The formation of PYP's signaling state is initiated by trans-cis isomerization of the p-coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is approximately 0.3. Using femtosecond visible pump/mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I0, both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I0-to-I1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.

How light-induced charge transfer accelerates the receptor-state recovery of photoactive yellow protein from its signaling state.

Stark (electroabsorption) spectra of the M100A mutant of photoactive yellow protein reveal that the neutral, cis cofactor of the pB intermediate undergoes a strikingly large change in the static dipole moment (|Deltamu| = 19 Debye) on photon absorption. The formation of this charge-separated species, in the excited state, precedes the cis --> trans isomerization of the pB cofactor and the regeneration of pG. The large |Deltamu|, reminiscent of that produced on the excitation of pG, we propose, induces twisting of the cis cofactor as a result of translocation of negative charge, from the hydroxyl oxygen, O1, toward the C7-C8 double bond. The biological significance of this photoinduced charge transfer reaction underlies the significantly faster regeneration of pG from pB in vitro, on the absorption of blue light.

From primary photochemistry to biological function in the blue-light photoreceptors PYP and AppA.

To properly respond to changes in fluency conditions, Nature has developed a variety of photosensors that modulate gene expression, enzyme activity and/or motility. Dedicated types have evolved, which can be classified in six families: rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins and BLUF-proteins. The photochemistry of the first three families is based on cis/trans isomerization of an ethylene bond. Surprisingly, the latter three all use flavin as their chromophore, but each with very different photochemistry. In this contribution we will discuss the molecular basis of signal generation in a xanthopsin (Photoactive Yellow Protein (PYP) from Halorhodospira halophila), a photoreceptor for negative phototaxis, and in a BLUF protein (AppA from Rhodobacter sphaeroides), a transcriptional anti-repressor. PYP is activated through trans/cis isomerization of the 7,8-vinyl bond of its 4-hydroxycinnamic acid chromophore. This initiates a photocycle with multiple intermediates, like pB, which is formed after intramolecular proton transfer. The negative charge thus formed in the interior of the protein triggers formation of a partially unfolded signaling state. For AppA much less is known about the underlying photochemistry. Available evidence suggests that it is based on a light-induced change in the hydrogen-bonding of its flavin chromophore and/or a change in hydrophobic stacking between the flavin and/or nearby aromatic amino acids like Y 21. A signaling state is formed within microseconds, which recovers with a rate of approximately 10(-3) s(-1). The change in conformation between receptor- and signaling-state in AppA, however, appear to be minute as compared to those in PYP. Here we review the underlying chemistry in the various steps of the photocycle of these two photoreceptor proteins and provide new data on their mechanism and function.

Stark spectroscopy on photoactive yellow protein, E46Q, and a nonisomerizing derivative, probes photo-induced charge motion.

The change in the electrostatic properties on excitation of the cofactor of wild-type photoactive yellow protein (WT-PYP) have been directly determined using Stark-effect spectroscopy. We find that, instantaneously on photon absorption, there is a large change in the permanent dipole moment, /Delta(-->)mu/, (26 Debye) and in the polarizability, (-)Deltaalpha, (1000 A(3)). We expect such a large degree of charge motion to have a significant impact on the photocycle that is associated with the important blue-light negative phototactic response of Halorhodospira halophila. Furthermore, changing E46 to Q in WT-PYP does not significantly alter its electrostatic properties, whereas, altering the chromophore to prevent it from undergoing trans-cis isomerization results in a significant diminution of /Delta(-->)mu/ and (-)Deltaalpha. We propose that the enormous charge motion that occurs on excitation of 4-hydroxycinnamyl thioester, the chromophore in WT-PYP, plays a crucial role in initiating the photocycle by translocation of the negative charge, localized on the phenolate oxygen in the ground state, across the chromophore. We hypothesize that this charge motion would consequently increase the flexibility of the thioester tail thereby decreasing the activation barrier for the rotation of this moiety in the excited state.

The role of the N-terminal domain of photoactive yellow protein in the transient partial unfolding during signalling state formation.

It is shown that the N-terminal domain of photoactive yellow protein (PYP), which appears relatively independently folded in the ground state of the protein, plays a key role in the transient unfolding during signalling state formation: genetic truncation of the N-terminal domain of PYP significantly decreases the extent of cooperativity of the titration curve that describes chromophore protonation in the ground state of PYP, which is in agreement with the notion that the N-terminal domain is linked through a hydrogen-bonding network with the chromophore-containing domain of the protein. Furthermore, deletion of the N-terminal domain completely abolishes the non-linearity of the Arrhenius plot of the rate of ground state recovery.