Structure of a photoactive rhodium complex intercalated into DNA

Intercalating complexes of rhodium(III) are strong photo-oxidants that promote DNA strand cleavage or electron transfer through the double helix. The 1.2 A resolution crystal structure of a sequence-specific rhodium intercalator bound to a DNA helix provides a rationale for the sequence specificity of rhodium intercalators. It also explains how intercalation in the center of an oligonucleotide modifies DNA conformation. The rhodium complex intercalates via the major groove where specific contacts are formed with the edges of the bases at the target site. The phi ligand is deeply inserted into the DNA base pair stack. The primary conformational change of the DNA is a doubling of the rise per residue, with no change in sugar pucker from B-form DNA. Based upon the five crystallographically independent views of an intercalated DNA helix observed in this structure, the intercalator may be considered as an additional base pair with specific functional groups positioned in the major groove.     

The fractionation of plant photoactive pigment-protein complexes I and II

The pigment-protein complexes enriched with Photosystem I (PPC-I) and Photosystem II (PPC-II) were obtained using sievorptive chromatography on DEAE-Sephadex column. Both types of complexes contain Chlorophyll a, β-carotene and minor quantities of Chl b. Red absorbance maxima are located at 676 nm and 673 nm for PPC-I and PPC-II, respectively. The degrees of reaction centre enrichment were measured by the method of differential spectrophotometry: PPC-I has one P-700 per 35 bulk Chl a molecules, PPC-II contains one P-680 per 18 bulk Chl a molecules. The yield of PPC-II is 7–10 times lower than that of PPC-I. After one chromatographic procedure the amount of P-680 in PPC-I preparation does not exceed 7% of that of P-700, the amount of P-700 in PPC-II preparation 2% of that of P-680. The product of PPC-II degradation was studied.     

Structure and dynamics of a tryptophanepeptide-polynucleotide complex

The binding of LysTrpLys to single stranded poly(A) was studied by measurements of fluorescence, UV-absorbance, electrodichroism and field jump relaxation. The van't Hoff enthalpy determined at constant degree of peptide protonation is -3.5 kcal/mol (delta S = 9 e.u.). The electrodichroism of bound tryptophane residues is negative; its absolute value decreases with increasing degree of binding theta. The magnitude of the dichroism at low theta indicates a preferential orientation of the tryptophane residues in the plane of the adenine bases, suggesting stacking of Trp with adenine bases. The overall degree of orientation decreases, however, to virtually zero at high theta. Relaxation measurements by low theta demonstrate the existence of two steps in the binding reaction of LysTrpLys to poly(A): a fast bimolecular step controlled by diffusion is followed by a slow intramolecular conversion with a forward rate of 1.5 x 10(5) s-1 and a backward rate of 2.7 x 10(3) s-1. The forward rate is close to that expected for an insertion reaction into stacked poly(A), yet the corresponding stability constant (approximately 55) is unexpectedly high.     

Reaction kinetics of electron phototransfer in an eosin-casein complex

The role of protein matrix in the process of charge photorespiration on the model of eosin-casein complex was studied. We have also studied the kinetics of the electron transfer reactions being photosensitized by free eosin and eosin sorbed on casein in the donor (cystein) - acceptor (methylviologen) system. When eosin is sorbed on protein a 10-fold increase of the probability relation of non-retrospective direct electron transfer to the restrospective one was observed.     

Antioxidant effects of carotenoids in a model pigment-protein complex

The effect of carotenoids on stability of model photosynthetic pigment-protein complexes subjected to chemical oxidation with hydrogen peroxide or potassium ferricyanide was investigated. The oxidation of carotenoid-less and carotenoid-containing complexes was conducted in the presence or absence of ascorbic acid. The progress of the reactions was monitored by use of absorption and fluorescence spectroscopy. Our results show that carotenoids may significantly enhance the stability of photosynthetic complexes against oxidation and their protective (antioxidant) effect depends on the type of the oxidant.     

Molecular arrangement of pigment-protein complex of photosystem 1

The circular dichroism (CD) method was applied to study the molecular organization of P700, antenna chlorophyll and protein of photosystem 1 complexes (CP1), isolated from chloroplasts under mild treatment with Triton X-100. Analysis of CD spectra and protein: chlorophyll: P700 ratios for CP1 complexes that were different in their chlorophyll content indicate that CP1 preparations can be considered as a mixture of CP1-RC, containing P700 (10–20%), and CP1-LH without P700 (80–90%). Both types of complexes contain approximately 25 chlorophyll molecules, and the destruction of their spatial organization with detergents represents a cooperative transition. The rate of chlorophyll destruction in CP1-LH is much higher than that in CP1-RC. In both complexes a 65 kDa polypeptide predominates, whose secondary structure (typical for / proteins) is stable to Triton X-100 and does not depends on the chlorophyll content. Chlorophyll seems to be grouped in clusters (5–7 molecules) in the hydrophobic cores of 2–3 parallel / domains of the 65 kDa protein. Only one of the clusters in CP1-RC includes P700; on P700 photooxidation the change of its interaction with the nearest pigment environment results in a complicated shape of the light-induced CD spectra.Abbreviations PS1 photosystem 1 - CP1 pigment-protein complex of PS1 - Chl chlorophyll a - CP1-140 CP1 with ratio Ch1:P700 140 - RC reaction center - LH light-harvesting pigment - CP1-RC CP1, containing P700 - CP1-LH CP1 without P700 (containing LH) - CD circular dichroism - SDS sodium dodecyl sulfate Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

Interaction of DNA with a novel photoactive platinum diimine complex

Interaction of DNA with a novel photoactive platinum diimine compound has been studied by electronic absorption spectra, fluorescence spectra and viscosity measurements. The red light-induced DNA cleavage activity of the platinum compound has also been studied by agarose gel electrophoresis. The results suggest that the platinum compound may interact with DNA by intercalation mode. When irradiated with red light, the platinum compound can generate singlet oxygen, resulting in cleavage of DNA.     

A diatom light-harvesting pigment-protein complex : purification and characterization

A light-harvesting pigment-protein complex was isolated from the diatom Phaeodactylum tricornutum using the zwitterionic detergent CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Detergent-solubilized membranes were fractionated by sucrose density gradient centrifugation into three components. The medium density fraction contained chlorophyll a, chlorophyll c, and fucoxanthin. This fraction was purified by DEAE-ion exchange chromatography, and contained chlorophyll a, chlorophyll c, and fucoxanthin in a molar ratio of 2.4:1.0:4.8. Fluorescence emission and excitation spectra of the isolated complex demonstrated that light energy absorbed by chlorophyll c and fucoxanthin was coupled to chlorophyll a fluorescence. Upon denaturation, the apoprotein yielded a polypeptide doublet at 17.5 to 18.0 kilodaltons which accounted for 30 to 40% of the toal membrane protein. These findings indicate that this pigment-protein complex is a major component of the diatom photosynthetic lammellae. The quantitative amino acid composition of the apoprotein was very similar to those reported for other membrane-bound pigment-protein complexes. Based on the protein to chlorophyll a ratio of 7700 grams protein per mole chlorophyll a for the complex, each apoprotein molecule contains, to the nearest integer, two chlorophyll a, one chlorophyll c, and five fucoxanthin molecules. Polyclonal antibodies raised against the 17.5 to 18.0 kilodaltons apoprotein showed a monospecific reaction with only the 17.5 to 18.0 protein zone from denatured P. tricornutum membranes as well as to the nondenatured pigment-protein complex. It appears that this complex is common to other diatom species.     

Structure and dynamics of the mammalian ribosomal pretranslocation complex

Although the structural core of the ribosome is conserved in all kingdoms of life, eukaryotic ribosomes are significantly larger and more complex than their bacterial counterparts. The extent to which these differences influence the molecular mechanism of translation remains elusive. Multiparticle cryo-electron microscopy and single-molecule FRET investigations of the mammalian pretranslocation complex reveal spontaneous, large-scale conformational changes, including an intersubunit rotation of the ribosomal subunits. Through structurally related processes, tRNA substrates oscillate between classical and at least two distinct hybrid configurations facilitated by localized changes in their L-shaped fold. Hybrid states are favored within the mammalian complex. However, classical tRNA positions can be restored by tRNA binding to the E site or by the eukaryotic-specific antibiotic and translocation inhibitor cycloheximide. These findings reveal critical distinctions in the structural and energetic features of bacterial and mammalian ribosomes, providing a mechanistic basis for divergent translation regulation strategies and species-specific antibiotic action.     

Ligand-dependent dynamics and intramolecular signaling in a PDZ domain

Allosteric communication is a fundamental process that proteins use to propagate signals from one site to functionally important distal sites. Although allostery is usually associated with multimeric proteins and enzymes, “long-range” communication may be a fundamental property of proteins. In some cases, communication occurs with minimal structural change. PDZ (post-synaptic density-95/discs large/zonula occludens-1) domains are small, protein-protein binding modules that can use multiple surfaces for docking diverse molecules. Furthermore, these domains have long-range energetic couplings that link the ligand-binding site to distal regions of the structure. Here, we show that allosteric behavior in a representative member of the PDZ domain family may be directly detected using side-chain methyl dynamics measurements. The changes in side-chain dynamics parameters in the second PDZ domain from the human tyrosine phosphatase 1E (hPTP1E) were determined upon binding a peptide target. Long-range dynamic effects were detected that correspond to previously observed pair-wise energetic couplings. These results provide one of the first experimental examples for the potential role of ps-ns timescale dynamics in propagating long-range signals within a protein, and reinforce the idea that dynamic fluctuations in proteins contribute to allosteric signal transduction.     

Isolation of a photoactive photosynthetic reaction center-core antenna complex from Heliobacillus mobilis

A photoactive reaction center-core antenna complex was isolated from the photosynthetic bacterium Heliobacillus mobilis by extraction of membranes with Deriphat 160c followed by differential centrifugation and sucrose density gradient ultracentrifugation. The purified complex contained a Mr 47,000 polypeptide(s) that bound both the primary donor (P800) and approximately 24 antenna bacteriochlorophylls g. Time-resolved fluorescence emission spectroscopy indicated that the antenna bacteriochlorophylls g are active in energy transfer to P800, exhibiting a decay time of 25 ps. The complex contained 1.4 menaquinones, 9 Fe, and 3 labile S2- per P800. The complex was photoactive with an exponential decay time of 14 ms for P800+ yet showed no EPR-detectable Fe-S center signal in the g less than or equal to 2.0 region, either by chemical reduction to -600 mV or by illumination of reduced samples. The complex is similar to photosystem I of oxygen-evolving photosynthetic systems in that both the primary donor and a core antenna are bound to the same pigment-protein complex.     

Molecular dynamics simulations of the photoactive protein nitrile hydratase

Nitrile hydratase (NHase) is an enzyme used in the industrial biotechnological production of acrylamide. The active site, which contains nonheme iron or noncorrin cobalt, is buried in the protein core at the interface of two domains, α and β. Hydrogen bonds between βArg-56 and αCys-114 sulfenic acid (αCEA114) are important to maintain the enzymatic activity. The enzyme may be inactivated by endogenous nitric oxide (NO) and activated by absorption of photons of wavelength λ < 630 nm. To explain the photosensitivity and to propose structural determinants of catalytic activity, differences in the dynamics of light-active and dark-inactive forms of NHase were investigated using molecular dynamics (MD) modeling. To this end, a new set of force field parameters for nonstandard NHase active sites have been developed. The dynamics of the photodissociated NO ligand in the enzyme channel was analyzed using the locally enhanced sampling method, as implemented in the MOIL MD package. A series of 1 ns trajectories of NHases shows that the protonation state of the active site affects the dynamics of the catalytic water and NO ligand close to the metal center. MD simulations support the catalytic mechanism in which a water molecule bound to the metal ion directly attacks the nitrile carbon.     

Polypeptide composition of the purified photosystem II pigment-protein complex from spinach.

The Photosystem II pigment-protein complex, the chlorophyll alpha-protein comprising the reaction center of Photosystem II, was prepared from EDTA-treated spinach chloroplasts by digitonin extraction, sucrose-gradient centrifugation, DEAE-cellulose column chromatography, and isoelectrofocussing on Ampholine. The dissociated pigment-protein complex exhibits two polypeptide subunits that migrate in SDS-polyacrylamide gel with electrophoretic mobilities corresponding to molecular weights of approximately 43,000 and 27,000. the chlorophyll was always found in the free pigment zone at the completion of the electrophoresis. Heat-treatment of the sample (100 degrees C, 90 s) for electrophoresis caused association of the two polypeptides into large aggregates. It is concluded that these two polypeptides, 43,000 and 27,000, are valid structural or functional components of Photosystem II pigment-protein complex.     

Polypeptide composition of the purified Photosystem II pigment-protein complex from spinach

The Photosystem II pigment-protein complex, the chlorophyll α-protein comprising the reaction center of Photosystem II, was prepared from EDTA-treated spinach chloroplasts by digitonin extraction, sucrose-gradient centrifugation, DEAE-cellulose column chromatography, and isoelectrofocussing on Ampholine.The dissociated pigment-protein complex exhibits two polypeptide subunits that migrate in SDS-polyacrylamide gel with electrophoretic mobilities corresponding to molecular weights of approximately 43 000 and 27 000. The chlorophyll was always found in the free pigment zone at the completion of the electrophoresis. Heat-treatment of the sample (100°C, 90 s) for electrophoresis caused association of the two polypeptides into large aggregates. It is concluded that these two polypeptides, 43 000 and 27 000, are valid structural or functional components of Photosystem II pigment-protein complex.     

Isolation and characterization of pigment-protein particles from the light-harvesting complex of Phaeodactylum tricornutum

The light-harvesting complex of the marine diatom Phaeodactylum tricornutum was fractionated into two large pigment-protein particles. One pigment-protein particle, which was contained in a yellow fraction, has a molecular weight, determined by gel filtration, of approx. 230 000 and can be dissociated in sodium dodecyl sulfate/mercaptoethanol solution to apopolypeptides of approx. 15 000. Characterization of particles with regard to molecular weights, subunits, protein and pigments suggests approx. 12 subunits per particle. The other pigment-protein particle, which was found in a green fraction, of approx. 95 000 molecular weight also reduces to apopolypeptide subunits of approx. 15 kDa. The relative molar proportions of chlorophyll a, chlorophyll c, fucoxanthin and total other accessory pigments in the former fraction are 3:1.3:6:2, whereas the proportions in the latter fraction are 5:1:3:1.     

Structure and dynamics of the quaternary hunchback mRNA translation repression complex

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.     

Backbone dynamics in an intramolecular prolylpeptide-SH3 complex from the diphtheria toxin repressor, DtxR

The diphtheria toxin repressor contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment and stabilizes the inactive state of the repressor. Upon activation of diphtheria toxin repressor (DtxR) by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active repressor. Here we investigate the dynamics of this intramolecular complex using backbone amide nuclear spin relaxation rates determined using NMR spectroscopy and molecular dynamics trajectories. The SH3 domain in the unbound and bound states showed typical dynamics in that the secondary structures were fairly ordered with high generalized order parameters and low effective correlation times, while residues in the loops connecting β-strands exhibited reduced generalized order parameters and required additional motional terms to adequately model the relaxation rates. Residues forming the Pr segment exhibited low-order parameters with internal rotational correlation times on the order of 0.6 ns-1 ns. Further analysis showed that the SH3 domain was rich in millisecond time scale motions while the Pr segment exhibited motions on the 100 μs time scale. Molecular dynamics simulations indicated structural rearrangements that may contribute to the observed relaxation rates and, together with the observed relaxation rate data, suggested that the Pr segment exhibits a binding ↔ unbinding equilibrium. The results here provide new insights into the nature of the intramolecular complex and provide a better understanding of the biological role of the SH3 domain in regulating DtxR activity.