Acceleration of catalysis in dihydrofolate reductase by transient, site-specific photothermal excitation.

We have studied the role of protein dynamics in chemical catalysis in the enzyme dihydrofolate reductase (DHFR), using a pump-probe method that employs pulsed-laser photothermal heating of a gold nanoparticle (AuNP) to directly excite a local region of the protein structure and transient absorbance to probe the effect on enzyme activity. Enzyme activity is accelerated by pulsed-laser excitation when the AuNP is attached close to a network of coupled motions in DHFR (on the FG loop, containing residues 116-132, or on a nearby alpha helix). No rate acceleration is observed when the AuNP is attached away from the network (distal mutant and His-tagged mutant) with pulsed excitation, or for any attachment site with continuous wave excitation. We interpret these results within an energy landscape model in which transient, site-specific addition of energy to the enzyme speeds up the search for reactive conformations by activating motions that facilitate this search.

A topologically distinct class of photolyases specific for UV lesions within single-stranded DNA.

Photolyases are ubiquitously occurring flavoproteins for catalyzing photo repair of UV-induced DNA damages. All photolyases described so far have a bilobal architecture with a C-terminal domain comprising flavin adenine dinucleotide (FAD) as catalytic cofactor and an N-terminal domain capable of harboring an additional antenna chromophore. Using sequence-similarity network analysis we discovered a novel subgroup of the photolyase/cryptochrome superfamily (PCSf), the NewPHLs. NewPHL occur in bacteria and have an inverted topology with an N-terminal catalytic domain and a C-terminal domain for sealing the FAD binding site from solvent access. By characterizing two NewPHL we show a photochemistry characteristic of other PCSf members as well as light-dependent repair of CPD lesions. Given their common specificity towards single-stranded DNA many bacterial species use NewPHL as a substitute for DASH-type photolyases. Given their simplified architecture and function we suggest that NewPHL are close to the evolutionary origin of the PCSf.

Low-Temperature Raman Spectroscopy of Halorhodopsin from Natronomonas pharaonis: Structural Discrimination of Blue-Shifted and Red-Shifted Photoproducts.

From the low-temperature absorption and Raman measurements of halorhodopsin from Natronomonas pharaonis (pHR), we observed that the two photoproducts were generated after exciting pHR at 80 K by green light. One photoproduct was the red-shifted K intermediate (pHRK) as the primary photointermediate for Cl- pumping, and the other was the blue-shifted one (pHRhypso), which was not involved in the Cl- pumping and thermally relaxed to the original unphotolyzed state by increasing temperature. The formation of these two kinds of photoproducts was previously reported for halorhodopsin from Halobacterium sarinarum [ Zimanyi et al. Biochemistry 1989 , 28 , 1656 ]. We found that the same took place in pHR, and we revealed the chromophore structures of the two photointermediates from their Raman spectra for the first time. pHRhypso had the distorted all-trans chromophore, while pHRK contained the distorted 13-cis form. The present results revealed that the structural analyses of pHRK carried out so far at ∼80 K potentially included a significant contribution from pHRhypso. pHRhypso was efficiently formed via the photoexcitation of pHRK, indicating that pHRhypso was likely a side product after photoexcitation of pHRK. The formation of pHRhypso suggested that the active site became tight in pHRK due to the slight movement of Cl-, and the back photoisomerization then produced the distorted all-trans chromophore in pHRhypso.

Engineering proteins for allosteric control by light or ligands.

Control of protein activity in living cells can reveal the role of spatiotemporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective in the interrogation of protein signaling, as they can replace endogenous proteins with minimal perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces an allosteric response comparable to the activity of native proteins. Here, we describe a detailed protocol to generate genetically encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, as well as experimental procedures to produce and test these analogs in vitro and in mammalian cell lines. We describe computational methods, based on crystal structures or homology models, to identify effective sites for insertion of either an engineered rapamycin-responsive (uniRapR) domain or the light-responsive light-oxygen-voltage 2 (LOV2) domain. The inserted domains allosterically regulate the active site, responding to rapamycin with irreversible activation, or to light with reversible inactivation at higher spatial and temporal resolution. These strategies have been successfully applied to catalytic domains of protein kinases, Rho family GTPases, and guanine exchange factors (GEFs), as well as the binding domain of a GEF Vav2. Computational tasks can be completed within a few hours, followed by 1-2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, GEF Vav2, and Rho GTPase Rac1 as examples.

Controlling protein conformation with light.

Optogenetics, genetically encoded engineering of proteins to respond to light, has enabled precise control of the timing and localization of protein activity in live cells and for specific cell types in animals. Light-sensitive ion channels have become well established tools in neurobiology, and a host of new methods have recently enabled the control of other diverse protein structures as well. This review focuses on approaches to switch proteins between physiologically relevant, naturally occurring conformations using light, accomplished by incorporating light-responsive engineered domains that sterically and allosterically control the active site.

Development of Visible-Light-Responsive RNA Scissors Based on a 10-23 DNAzyme.

The 10-23 DNAzyme is an artificially developed functional oligonucleotide that can cleave RNA in a sequence-specific manner. In this study, we designed a new photo-driven DNAzyme incorporating a photoresponsive DNA overhang complementary to the catalytic core region. The photoresponsive overhang region of the DNAzyme included either azobenzene components (Azos) or 2,6-dimethyl-4-(methylthio)azobenzene units (SDM-Azos) each attached to a d-threoninol linker. When the Azos or SDM-Azos were in the trans form, the photoresponsive DNA overhang hybridized with the DNAzyme, and the RNA cleavage activity was suppressed. cis Isomerization of Azos or SDM-Azos, induced by 365 or 400 nm light, respectively, destabilized the duplex between the photoresponsive overhang and the catalytic core, and the DNAzyme recovered RNA cleavage activity. Reversible photoswitching of the DNAzyme activity was achieved by use of specific light irradiation. Further, light-dependent photoswitching of protein expression in the presence of the DNAzyme was demonstrated. Thus, this photo-driven DNAzyme has potential for application as a photocontrolled gene silencing system and a photoactivatable gene expression system.

HDAC8 Substrates Identified by Genetically Encoded Active Site Photocrosslinking.

The histone deacetylase family comprises 18 enzymes that catalyze deacetylation of acetylated lysine residues; however, the specificity and substrate profile of each isozyme remains largely unknown. Due to transient enzyme-substrate interactions, conventional co-immunoprecipitation methods frequently fail to identify enzyme-specific substrates. Additionally, compensatory mechanisms often limit the ability of knockdown or chemical inhibition studies to achieve significant fold changes observed by acetylation proteomics methods. Furthermore, measured alterations do not guarantee a direct link between enzyme and substrate. Here we present a chemical crosslinking strategy that incorporates a photoreactive, non-natural amino acid, p-benzoyl-l-phenylalanine, into various positions of the structurally characterized isozyme histone deacetylase 8 (HDAC8). After covalent capture, co-immunoprecipitation, and mass spectrometric analysis, we identified a subset of HDAC8 substrates from human cell lysates, which were further validated for catalytic turnover. Overall, this chemical crosslinking approach identified novel HDAC8-specific substrates with high catalytic efficiency, thus presenting a general strategy for unbiased deacetylase substrate discovery.

Radiation damage at the active site of human alanine:glyoxylate aminotransferase reveals that the cofactor position is finely tuned during catalysis.

The alanine:glyoxylate aminotransferase (AGT), a hepatocyte-specific pyridoxal-5'-phosphate (PLP) dependent enzyme, transaminates L-alanine and glyoxylate to glycine and pyruvate, thus detoxifying glyoxylate and preventing pathological oxalate precipitation in tissues. In the widely accepted catalytic mechanism of the aminotransferase family, the lysine binding to PLP acts as a catalyst in the stepwise 1,3-proton transfer, interconverting the external aldimine to ketimine. This step requires protonation by a conserved aspartate of the pyridine nitrogen of PLP to enhance its ability to stabilize the carbanionic intermediate. The aspartate residue is also responsible for a significant geometrical distortion of the internal aldimine, crucial for catalysis. We present the structure of human AGT in which complete X-ray photoreduction of the Schiff base has occurred. This result, together with two crystal structures of the conserved aspartate pathogenic variant (D183N) and the molecular modeling of the transaldimination step, led us to propose that an interplay of opposite forces, which we named spring mechanism, finely tunes PLP geometry during catalysis and is essential to move the external aldimine in the correct position in order for the 1,3-proton transfer to occur.

Proton transfer reactions in the red light-activatable channelrhodopsin variant ReaChR and their relevance for its function.

Channelrhodopsins (ChRs) are light-gated ion channels widely used for activating selected cells in large cellular networks. ChR variants with a red-shifted absorption maximum, such as the modified Volvox carteri ChR1 red-activatable channelrhodopsin ("ReaChR," λmax = 527 nm), are of particular interest because longer wavelengths allow optical excitation of cells in deeper layers of organic tissue. In all ChRs investigated so far, proton transfer reactions and hydrogen bond changes are crucial for the formation of the ion-conducting pore and the selectivity for protons versus cations, such as Na+, K+, and Ca2+ (1). By using a combination of electrophysiological measurements and UV-visible and FTIR spectroscopy, we characterized the proton transfer events in the photocycle of ReaChR and describe their relevance for its function. 1) The central gate residue Glu130 (Glu90 in Chlamydomonas reinhardtii (Cr) ChR2) (i) undergoes a hydrogen bond change in D → K transition and (ii) deprotonates in K → M transition. Its negative charge in the open state is decisive for proton selectivity. 2) The counter-ion Asp293 (Asp253 in CrChR2) receives the retinal Schiff base proton during M-state formation. Starting from M, a photocycle branching occurs involving (i) a direct M → D transition and (ii) formation of late photointermediates N and O. 3) The DC pair residue Asp196 (Asp156 in CrChR2) deprotonates in N → O transition. Interestingly, the D196N mutation increases 15-syn-retinal at the expense of 15-anti, which is the predominant isomer in the wild type, and abolishes the peak current in electrophysiological measurements. This suggests that the peak current is formed by 15-anti species, whereas 15-syn species contribute only to the stationary current.

Structural study of the X-ray-induced enzymatic reduction of molecular oxygen to water by Steccherinum murashkinskyi laccase: insights into the reaction mechanism.

The laccase from Steccherinum murashkinskyi is a member of the large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates, accompanied by the reduction of dioxygen to water. The reducing properties of X-ray radiation and the high quality of the laccase crystals allow the study of the catalytic reduction of dioxygen to water directly in a crystal. A series of diffraction data sets with increasing absorbed radiation dose were collected from a single crystal of Steccherinum murashkinskyi laccase at 1.35 Šresolution. Changes in the active-site structure associated with the reduction of molecular oxygen to water on increasing the absorbed dose of ionizing radiation were detected. The structures in the series are mixtures of different states of the enzyme-substrate complex. Nevertheless, it was possible to interpret these structures as complexes of various oxygen ligands with copper ions in different oxidation states. The results allowed the mechanism of oxygen reduction catalyzed by laccases to be refined.

Effects of microwaves (900 MHz) on peroxidase systems: A comparison between lactoperoxidase and horseradish peroxidase.

This work shows the effects of exposure to an electromagnetic field at 900 MHz on the catalytic activity of the enzymes lactoperoxidase (LPO) and horseradish peroxidase (HRP). Experimental evidence that irradiation causes conformational changes of the active sites and influences the formation and stability of the intermediate free radicals is documented by measurements of enzyme kinetics, circular dichroism spectroscopy (CD) and cyclic voltammetry.

Superoxide reductase from Giardia intestinalis: structural characterization of the first SOR from a eukaryotic organism shows an iron centre that is highly sensitive to photoreduction.

Superoxide reductase (SOR), which is commonly found in prokaryotic organisms, affords protection from oxidative stress by reducing the superoxide anion to hydrogen peroxide. The reaction is catalyzed at the iron centre, which is highly conserved among the prokaryotic SORs structurally characterized to date. Reported here is the first structure of an SOR from a eukaryotic organism, the protozoan parasite Giardia intestinalis (GiSOR), which was solved at 2.0 Å resolution. By collecting several diffraction data sets at 100 K from the same flash-cooled protein crystal using synchrotron X-ray radiation, photoreduction of the iron centre was observed. Reduction was monitored using an online UV-visible microspectrophotometer, following the decay of the 647 nm absorption band characteristic of the iron site in the glutamate-bound, oxidized state. Similarly to other 1Fe-SORs structurally characterized to date, the enzyme displays a tetrameric quaternary-structure arrangement. As a distinctive feature, the N-terminal loop of the protein, containing the characteristic EKHxP motif, revealed an unusually high flexibility regardless of the iron redox state. At variance with previous evidence collected by X-ray crystallography and Fourier transform infrared spectroscopy of prokaryotic SORs, iron reduction did not lead to dissociation of glutamate from the catalytic metal or other structural changes; however, the glutamate ligand underwent X-ray-induced chemical changes, revealing high sensitivity of the GiSOR active site to X-ray radiation damage.

Structural changes caused by radiation-induced reduction and radiolysis: the effect of X-ray absorbed dose in a fungal multicopper oxidase.

X-ray radiation induces two main effects at metal centres contained in protein crystals: radiation-induced reduction and radiolysis and a resulting decrease in metal occupancy. In blue multicopper oxidases (BMCOs), the geometry of the active centres and the metal-to-ligand distances change depending on the oxidation states of the Cu atoms, suggesting that these alterations are catalytically relevant to the binding, activation and reduction of O(2). In this work, the X-ray-determined three-dimensional structure of laccase from the basidiomycete Coriolopsis gallica (Cg L), a high catalytic potential BMCO, is described. By combining spectroscopic techniques (UV-Vis, EPR and XAS) and X-ray crystallography, structural changes at and around the active copper centres were related to pH and absorbed X-ray dose (energy deposited per unit mass). Depletion of two of the four active Cu atoms as well as low occupancies of the remaining Cu atoms, together with different conformations of the metal centres, were observed at both acidic pH and high absorbed dose, correlating with more reduced states of the active coppers. These observations provide additional evidence to support the role of flexibility of copper sites during O(2) reduction. This study supports previous observations indicating that interpretations regarding redox state and metal coordination need to take radiation effects explicitly into account.

Coupling between the BLUF and EAL domains in the blue light-regulated phosphodiesterase BlrP1.

The first biochemical and structural characterization of the full-length active photoreceptor BlrP1 from Klebsiella pneumoniae was recently reported by Barends et al. [Nature 459:1015-1018, (2009)]. The light-regulated catalytic function of its C-terminal c-di-guanosine monophosphate phosphodiesterase, the EAL (Glu-Ala-Leu) domain, is activated by the N-terminal sensor of blue light using the flavin adenine dinucleotide (BLUF) domain. We performed molecular dynamics simulations on the dimeric BlrP1 protein in order to examine the coupling regions that are presumably involved in transmitting light-induced structural changes which occur in the BLUF domain to the EAL domain. According to the results of simulations and an analysis of the hydrogen bonding between the respective polypeptide chains, the region containing the site on the α3α4 loop of BLUF is responsible for communication between the photosensing and catalytic domains in the dimeric BlrP1 protein.

Phytochrome three-dimensional structures and functions.

The complete three-dimensional sensory module structures of the Pr ground state of Synechocystis 6803 Cph1 and the unusual Pfr ground state of the bacteriophytochrome PaBphP (PDB codes 2VEA and 3C2W respectively) have now been solved, revealing an asymmetrical dumbbell form made up of a PAS (Period/ARNT/Singleminded)-GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) bidomain carrying the chromophore and the smaller PHY (phytochrome-specific) domain. The PHY domain is structurally related to the GAF family, but carries an unusual tongue-like structure which contacts the larger lobe to seal the chromophore pocket. In 2VEA, the tongue makes intimate contact with the helical N-terminus; both the N-terminus and the tongue structures are quite different in 3C2W. As expected, the structures reveal ZZZssa and ZZEssa chromophore conformations in 2VEA and 3C2W respectively, associated with tautomeric differences in several nearby tyrosine residues. Two salt bridges on opposite sides of the chromophore, as well as the associations of the C-ring propionates also differ. It is still unclear, however, which of these structural differences are associated with bacteriophytochromes compared with Cph1 and plant-type phytochromes, the unusual 3C2W Pfr ground state functionality compared with the Pr ground state or the Pr compared with Pfr photoisomerism. To access the latter unambiguously, both Pr and Pfr structures of the same molecule are required. New solid-phase NMR data for Cph1 in the Pr, Pfr and freeze-trapped intermediate states reveal unexpected changes in the chromophore during Pfr-->Pr photoconversion. These, together with our efforts to solve the three-dimensional structure of a complete phytochrome molecule are also described.

Conformational changes in an ultrafast light-driven enzyme determine catalytic activity.

The role of conformational changes in explaining the huge catalytic power of enzymes is currently one of the most challenging questions in biology. Although it is now widely regarded that enzymes modulate reaction rates by means of short- and long-range protein motions, it is almost impossible to distinguish between conformational changes and catalysis. We have solved this problem using the chlorophyll biosynthetic enzyme NADPH:protochlorophyllide (Pchlide) oxidoreductase, which catalyses a unique light-driven reaction involving hydride and proton transfers. Here we report that prior excitation of the enzyme-substrate complex with a laser pulse induces a more favourable conformation of the active site, enabling the coupled hydride and proton transfer reactions to occur. This effect, which is triggered during the Pchlide excited-state lifetime and persists on a long timescale, switches the enzyme into an active state characterized by a high rate and quantum yield of formation of a catalytic intermediate. The corresponding spectral changes in the mid-infrared following the absorption of one photon reveal significant conformational changes in the enzyme, illustrating the importance of flexibility and dynamics in the structure of enzymes for their function.

Binding of the DNA-dependent protein kinase catalytic subunit to Holliday junctions.

DNA-PK (DNA-dependent protein kinase) is a double-strand break sensor involved in DNA repair and signal transduction. In the present study, we constructed site-directed cross-linking probes to explore the range of DNA discontinuities that are recognized by DNA-PK(CS) (DNA-PK catalytic subunit). A comparison between different substrate architectures showed that DNA-PK(CS) associates preferentially with the crossover region of synthetic Holliday junctions. This interaction with four-way junctions was preserved when biotin-streptavidin complexes were assembled at the termini to exclude the binding of Ku proteins. The association of DNA-PK(CS) with Holliday junctions was salt-labile even in the presence of Ku proteins, but this interaction could be stabilized when the DNA probes were incubated with the endogenous enzyme in nuclear extracts of human cells. Cross-linking of the endogenous enzyme in cellular extracts also demonstrated that DNA-PK(CS) binds to DNA ends and four-way junctions with similar affinities in the context of a nuclear protein environment. Kinase assays using p53 proteins as a substrate showed that, in association with four-way structures, DNA-PK(CS) adopts an active conformation different from that in the complex with linear DNA. Our results are consistent with a structure-specific, but Ku- and DNA end-independent, recruitment of DNA-PK(CS) to Holliday junction intermediates. This observation suggests an unexpected functional link between the two main pathways that are responsible for the repair of DNA double-strand breaks in mammalian cells.

Poly(ADP-ribose) polymerase, a major determinant of early cell response to ionizing radiation.

PURPOSE: To determine whether DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP-1) are involved in eliciting the rapid fluctuations of radiosensitivity that have been observed when cells are exposed to short pulses of ionizing radiation. MATERIALS AND METHODS: The effect of DNA-PK and PARP-1 inhibitors on the survival of cells to split-dose irradiation was investigated using Chinese hamster V79 fibroblasts and human carcinoma SQ-20B cells. The responses of PARP-1 proficient and PARP-1 knockout mouse 3T3 fibroblasts were compared in a similar split-dose assay. RESULTS: Inactivation of DNA-PK by wortmannin potentiated radiation-induced cell kill but it did not alter the oscillatory, W-shaped pattern of early radiation response. In contrast, oscillatory radiation response was abolished by 3-aminobenzamide, a reversible inhibitor of enzymes containing a PARP catalytic domain. The oscillatory response was also lacking in PARP-1 knockout mouse 3T3 fibroblasts. CONCLUSION: The results show that PARP-1 plays a key role in the earliest steps of cell response to ionizing radiation with clonogenic ability or growth as endpoint. It is hypothesized that rapid poly(ADP-ribosylation) of target proteins, or recruitment of repair proteins by activated PARP-1 at the sites of DNA damage, bring about rapid chromatin remodelling that may affect the incidence of chromosomal damage upon re-irradiation.