Georgios Psakis

Two ground state isoforms and a chromophore D-ring photoflip triggering extensive intramolecular changes in a canonical phytochrome

Phytochrome photoreceptors mediate light responses in plants and in many microorganisms. Here we report studies using 1H–13C magic-angle spinning NMR spectroscopy of the sensor module of cyanobacterial phytochrome Cph1. Two isoforms of the red-light absorbing Pr ground state are identified. Conclusive evidence that photoisomerization occurs at the C15-methine bridge leading to a β-facial disposition of the ring D is presented. In the far-red-light absorbing Pfr state, strong hydrogen-bonding interactions of the D-ring carbonyl group to Tyr-263 and of N24 to Asp-207 hold the chromophore in a tensed conformation. Signaling is triggered when Asp-207 is released from its salt bridge to Arg-472, probably inducing conformational changes in the tongue region. A second signal route is initiated by partner swapping of the B-ring propionate between Arg-254 and Arg-222.

Spectroscopy and a High-Resolution Crystal Structure of Tyr263 Mutants of Cyanobacterial Phytochrome Cph1.

Phytochromes are biliprotein photoreceptors that can be photoswitched between red-light-absorbing state (Pr) and far-red-light-absorbing state (Pfr). Although three-dimensional structures of both states have been reported, the photoconversion and intramolecular signaling mechanisms are still unclear. Here, we report UV-Vis absorbance, fluorescence and CD spectroscopy along with various photochemical parameters of the wild type and Y263F, Y263H and Y263S mutants of the Cph1 photosensory module, as well as a 2.0-Å-resolution crystal structure of the Y263F mutant in its Pr ground state. Although Y263 is conserved, we show that the aromatic character but not the hydroxyl group of Y263 is important for Pfr formation. The crystal structure of the Y263F mutant (Protein Data Bank ID: 3ZQ5) reaffirms the ZZZssa chromophore configuration and provides a detailed picture of its binding pocket, particularly conformational heterogeneity around the chromophore. Comparison with other phytochrome structures reveals differences in the relative position of the PHY (phytochrome specific) domain and the interaction of the tongue with the extreme N-terminus. Our data support the notion that native phytochromes in their Pr state are structurally heterogeneous.

Flexibility of the N-Terminal mVDAC1 Segment Controls the Channel’s Gating Behavior

Since the solution of the molecular structures of members of the voltage dependent anion channels (VDACs), the N-terminal α-helix has been the main focus of attention, since its strategic location, in combination with its putative conformational flexibility, could define or control the channel’s gating characteristics. Through engineering of two double-cysteine mVDAC1 variants we achieved fixing of the N-terminal segment at the bottom and midpoint of the pore. Whilst cross-linking at the midpoint resulted in the channel remaining constitutively open, cross-linking at the base resulted in an “asymmetric” gating behavior, with closure only at one electric field´s orientation depending on the channel’s orientation in the lipid bilayer. Additionally, and while the native channel adopts several well-defined closed states (S1 and S2), the cross-linked variants showed upon closure a clear preference for the S2 state. With native-channel characteristics restored following reduction of the cysteines, it is evident that the conformational flexibility of the N-terminal segment plays indeed a major part in the control of the channel’s gating behavior.

The D-ring, not the A-ring, Rotates in Synechococcus OS-B' Phytochrome

Background: Phytochrome photoreceptors are activated by light-induced isomerization of the chromophore cofactor. Results: Photoactivation of Synechococcus OS-B′ phytochrome breaks an unusual chromophore D-ring hydrogen bond, whereas only subtle changes occur at the A-ring linkage to the protein. Conclusion: Activation arises from a photoflip of a strongly tilted D-ring. Significance: The hypothesis that the A-ring rotates upon photon absorption is wrong. Phytochrome photoreceptors in plants and microorganisms switch photochromically between two states, controlling numerous important biological processes. Although this phototransformation is generally considered to involve rotation of ring D of the tetrapyrrole chromophore, Ulijasz et al. (Ulijasz, A. T., Cornilescu, G., Cornilescu, C. C., Zhang, J., Rivera, M., Markley, J. L., and Vierstra, R. D. (2010) Nature 463, 250–254) proposed that the A-ring rotates instead. Here, we apply magic angle spinning NMR to the two parent states following studies of the 23-kDa GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domain fragment of phytochrome from Synechococcus OS-B′. Major changes occur at the A-ring covalent linkage to the protein as well as at the protein residue contact of ring D. Conserved contacts associated with the A-ring nitrogen rule out an A-ring photoflip, whereas loss of contact of the D-ring nitrogen to the protein implies movement of ring D. Although none of the methine bridges showed a chemical shift change comparable with those characteristic of the D-ring photoflip in canonical phytochromes, denaturation experiments showed conclusively that the same occurs in Synechococcus OS-B′ phytochrome upon photoconversion. The results are consistent with the D-ring being strongly tilted in both states and the C15=C16 double bond undergoing a Z/E isomerization upon light absorption. More subtle changes are associated with the A-ring linkage to the protein. Our findings thus disprove A-ring rotation and are discussed in relation to the position of the D-ring, photoisomerization, and photochromicity in the phytochrome family.

Voltage-dependent anion channels: the wizard of the mitochondrial outer membrane.

Abstract Voltage dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane. Although they are essential in metabolite exchange, cell defense and apoptosis, the molecular mechanism of these VDAC-mediated processes remains elusive. Here we review recent progress in terms of VDACs’ structure and regulation, with a special focus on the molecular aspects of gating and the interaction with effector proteins.

AcrB et al.: Obstinate contaminants in a picogram scale. One more bottleneck in the membrane protein structure pipeline

Heterologous expression of integral membrane proteins from Helicobacter pylori 26695 in Escherichia coli enabled the identification of 17 candidates for purification and subsequent crystallization. 45% of the purified proteins were contaminated with what was later identified as the multidrug efflux pump (AcrB)of E. coli, and 17% with the succinate dehydrogenase. While additional purification steps ensured removal of succinate dehydrogenase, they failed to remove AcrB completely, leaving picogram amounts present infractions intended for 3D-crystallization. Two of these targets, the Na+ dependent D-glucose/D-galactose transporter (GluP-HP1174) and the carbon starvation protein A (CstA-HP1168), produced small crystals(<40 lm). Crystals from the GluP preparation diffracted to 4.2 A resolution and belonged to the rhombohedral space group H32. Subsequent molecular replacement proved that these crystals were derived from a contaminant, the efflux transporter AcrB. This unexpected crystallization of AcrB from picogram amounts was observed in six new conditions. The systematic occurrence of AcrB in membrane preparations stems from the upregulation of its transcription in response to the stress induced by the expression of a selected target. This, along with its tendency to crystallize in the picogram scale, poses a serious concern in membrane protein expression using heterologous hosts harbouring AcrB.

NMR Spectroscopic Investigation of Mobility and Hydrogen Bonding of the Chromophore in the Binding Pocket of Phytochrome Proteins

For a complete understanding of the light reception of phytochrome proteins, a detailed study of the structure and dynamics of the binding pocket at atomic resolution is required. Structures from X-ray crystallography and NMR spectroscopy are available and have been able to provide a picture of the binding pocket. NMR spectroscopy has, in addition, shown that the chromophore exhibits noticeable dynamics in the binding pocket of the cyanobacterial phytochrome Cph1. Herein, NMR spectroscopy is used to investigate further the mobility of the chromophore by analyzing the line widths of the resonances of the chromophore in various environments, in particular other protein environments. It is shown that the chromophore exhibits a different mobility in the binding pocket of the bacterial phytochrome Agp1 than in that of the cyanobacterial phytochrome Cph1. Finally, it is shown that NMR spectroscopy is capable of detecting hydrogen bonds in the binding pocket of phytochromes by observing slow exchange of protons in the amino acid side chains.

Expression screening of integral membrane proteins from Helicobacter pylori 26695

The efficiency of Helicobacter pylori as a mucosal pathogen is caused by unique soluble and integral membrane proteins, which allow its survival at acidic pH and successful colonization of the gastric environment. With about one‐fourth of the H. pyloris proteome comprising integral membrane proteins, the need for solution of their three‐dimensional (3D) structures becomes persistent as it can potentially drive the generation of more effective drugs. This study presents a medium‐throughput approach for cloning and expression screening of integral membrane proteins from H. pylori (26695) using Escherichia coli as the expression host. One‐hundred sixteen H. pylori targets were cloned into two different vector systems and heterologously expressed in E. coli. Eighty‐four percent of these proteins displayed medium to high expression. No clear‐cut correlation was found between expression levels and number of putative transmembrane spans, predicted functionality, and molecular mass. Nonetheless, expression of transporters and hypothetical proteins ≤40 kDa with two to four transmembrane spans displayed generally high expression levels. To statistically strengthen the quality of the data from the medium‐throughput approach, a comparison with data derived from robotic‐based methodologies was conducted. Optimization of expression and solubilization conditions for selected targets was also performed. Seventeen targets have been purified and subjected to crystallization so far. Eighteen percent of these targets (2/17) produced crystals under specific sets of crystallization conditions.

Structure-Based Engineering of a Minimal Porin Reveals Loop-Independent Channel Closure

Porins, like outer membrane protein G (OmpG) of Escherichia coli, are ideal templates among ion channels for protein and chemical engineering because of their robustness and simple architecture. OmpG shows fast transitions between open and closed states, which were attributed to loop 6 (L6). As flickering limits single-channel-based applications, we pruned L6 by either 8 or 12 amino acids. While the open probabilities of both L6 variants resemble that of native OmpG, their gating frequencies were reduced by 63 and 81%, respectively. Using the 3.2 Å structure of the shorter L6 variant in the open state, we engineered a minimal porin (220 amino acids), where all remaining extramembranous loops were truncated. Unexpectedly, this minimized porin still exhibited gating, but it was 5-fold less frequent than in OmpG. The residual gating of the minimal pore is hence independent of L6 rearrangements and involves narrowing of the ion conductance pathway most probably driven by global stretching-flexing deformations of the membrane-embedded β-barrel.

On the collective nature of phytochrome photoactivation

The red/far-red-sensing biological photoreceptor phytochrome is a paradigmatic two-state signaling system. The two thermally stable states are interconverted via a photoreaction of the covalently bound tetrapyrrole chromophore. Applying recently developed solid-state nuclear magnetic resonance, we study both the chromophore and its protein pocket in the Pr (red-absorbing) and Pfr (far-red-absorbing) states. The observations show that the phototransformation combines local chemical reactions with a mesoscopic transition of order. Both the chromophore and its binding pocket are quasi-liquid and disordered in Pr, yet quasi-solid and ordered in Pfr. Possible biochemical implications are discussed.