RNAs often exhibit a high degree of conformational dynamics and heterogeneity, leading to a rugged energy landscape. However, the roles of conformational heterogeneity and rapid dynamics in molecular recognition or RNA function have not been extensively elucidated. Ultrafast time-resolved fluorescence spectroscopic experiments were used here to probe picosecond dynamics of the theophylline-binding RNA aptamer. These studies showed that multiple conformations are populated in the free RNA indicating that this aptamer employs a conformational capture mechanism for ligand binding. The base on residue 27 in an internal loop exists in at least three conformational states in the free RNA, including binding competent and incompetent states that have distinct fluorescence decay signatures indicating different base stacking interactions. Picosecond dynamics were also detected by anisotropy experiments, where these motions indicate additional dynamics for base 27. The picosecond data show that theophylline binding shifts the equilibrium for conformations of base 27 from primarily stacked in the free RNA to mostly unstacked in the RNA-theophylline complex, as observed in the previous NMR structure. In contrast, base 10 in a second internal loop is mostly pre-organized in the free RNA, consistent with it being stacked between G11 and G25, as is observed in the bound state. Picosecond dynamics were also measured on a modified aptamer that binds with higher affinity to 3-methylxanthine than theophylline. The modified aptamer shows less heterogeneity in the aptamer-3-methylxanthine complex than what is observed in the theophylline aptamer-theophylline complex.

NPRA is a non-covalent homodimeric receptor, composed of an extracellular domain (ECD) with a ligand-binding site, a single transmembrane domain (TM) and an intracellular domain (ICD) exhibiting guanylyl cyclase activity. NPRA activation by atrial natriuretic peptide (ANP) leads to cGMP production, which plays important roles in cardiovascular homeostasis. Initial studies have shown that activation of NPRA involves a conformational change of the juxtamembrane domain (JM). However, crystallographic study of the soluble ECD of NPRA has failed to document JM structure, and the conformational change involved in transmembrane signal transduction is still unknown. To analyse this conformational change, we first sequentially substituted nine amino acids of JM by a cysteine residue. By studying the mutant's capacity to form ANP-induced or constitutive covalent disulfide dimers, we evaluated the relative proximity of JM residues, before and after NPRA activation. These results obtained with full-length receptor demonstrate a high proximity of specific JM residues and are in disagreement with crystallography data. We also tested the hypothesis that signal transduction involves a TM rotation mechanism leading to ICD activation. By introducing 1 to 5 alanine residues in the TM alpha-helix, we show that a TM rotation of 40 degrees leads to constitutive NPRA activation. We finally studied the role of the TM in NPRA dimerization. By using the ToxR system, we demonstrate that the last JM residues are required to stabilize the TM dimer. Using these experimental data, we generated a new molecular model illustrating the active conformation of NPRA, where JM and TM are depicted.

In archaea and eukaryotes, the nascent polypeptide-associated complex (NAC) is one of the cytosolic chaperones that contact the nascent polypeptide chains as they emerge from the ribosome, and assists in post-translational processes. The eukaryotic NAC is a heterodimer, and its two subunits form a stable complex through a dimerizing domain called NAC domain. In addition to acting as protein translation chaperone, the NAC subunits also function individually in transcriptional regulation. Here we report the crystal structure of the human NAC domain, which reveals the manner of human NAC dimerization. Based on the structure, we identified a region in the NAC domain of the human NAC alpha-subunit as a new nucleic acid-binding region, which is blocked from binding nucleic acids in the heterodimeric complex by a helix region in the beta-subunit.

Transcription elongation is regulated by the cellular protein Hexim1, which inhibits phosphorylation of the RNA polymerase II by its interaction with the positive transcription elongation factor P-TEFb. Hexim1 binds directly to Cyclin T1 of P-TEFb with its coiled coil domain that is subdivided into a highly polar N-terminal segment containing non-conservative residues in the dimer interface and a C-terminal segment with evolutionarily conserved sequence composition. Here we show that the non-canonical sequence composition of the first coiled coil segment is required for the interaction with Cyclin T1 while the second segment keeps the Cyclin T-binding domain dimeric upon binding. Both coiled coil segments exhibit distinct melting points as shown by heat denaturation experiments using circular dichroism spectroscopy. Deletion of the central stammer motif (316-318) leads to a single denaturation reaction, suggesting formation of a continuous coiled coil. Mutation of non-canonical coiled coil residues K284 and Y291 to valines in the dimer interface of the first segment only slightly increases its stability. Concomitantly, deletion of the stammer but not the double point mutation led to a reduced affinity to Cyclin T1 as shown by isothermal titration calorimetry. Moreover, Cyclin T1 bound Hexim1 in a 1:2 stoichiometry, whereas truncation of the C-terminal coiled coil led to an equimolar complex formation. These observations suggest that binding to Cyclin T1 induces an asymmetry or sterical hindrance in the first coiled coil segment of dimeric Hexim1 that disallows formation of a 2:2 complex as further supported by analytical ultracentrifugation and cross-linking experiments.

Genetic regulation by non-coding RNA elements such as microRNA and small interfering RNA (siRNA) involves hybridization of a short single-stranded RNA with a complementary segment in a target mRNA. The physical basis of the hybridization process between the structured nucleic acids is not well understood primarily owing to the lack of information on the transition state structure. Here we use transition-state theory, inspired by Phi-value analysis in protein folding studies, to provide quantitative analysis of the relationship between changes in the secondary structure stability and the activation free energy. Time-course monitoring of the hybridization reaction was performed under pseudo-steady-state conditions using a single fluorophore. The Phi-value analysis indicates that the native secondary structure remains intact in the transition state. The native-like transition state was confirmed by examining salt dependence of the hybridization kinetics, indicating that the number of sodium ions associated with the transition state was not substantially affected by changes in the native secondary structure. These results propose that hybridization between structured nucleic acids undergoes a transition state leading to formation of a nucleation complex and then is followed by sequential displacement of pre-existing base pairings involving successive small energy barriers. The proposed mechanism might provide a new insight into physical processes during small RNA-mediated gene silencing, which is essential to selection of a target mRNA segment for siRNA design.

ABCG5 and ABCG8 are half-size ABC transporters that function as heterodimers (ABCG5/G8) to reduce sterol absorption in the intestines and increase sterol excretion from the liver. Previous studies demonstrated that bile acids increased ABCG5/G8 specific cholesterol efflux in cell models. In this study we tested the effects of bile acids on ATP hydrolysis in P. pastoris purified ABCG5/G8 and found that they stimulated hydrolysis ~20 fold in wild-type ABCG5/G8, but not in a hydrolysis deficient mutant. Non-conjugated cholate supported the highest ATPase activity in ABCG5/G8 (256 +/- 9 nmol/min/mg). ATP hydrolysis was also stimulated by other conjugated bile acids and a mixture of bile acids resembling human bile with activities ranging from 129 +/- 4 to 147 +/- 14 nmol/min/mg. The kinetic parameters, inhibitor profiles, and lipid requirements of bile acid stimulated ATP hydrolysis were characterized. Cholate stimulated ATP hydrolysis was maximal at concentrations of >/= 10 mM MgATP, and had a relatively high KM (MgATP) of ~ 1 mM. Orthovanadate, BeFx, and AlFx effectively inhibited ABCG5/G8 at concentrations of 1 mM. Various lipid mixtures supported bile acid-stimulated ATP hydrolysis, which increased when cholesterol was present. The data demonstrate that bile acids together with lipids and cholesterol increase ATP hydrolysis in purified ABCG5/G8. Bile acids may promote an active conformation of purified ABCG5/G8 either by global stabilization of the transporter or by binding to a specific site on ABCG5/G8.

Amyloid deposits, composed primarily of the 37-residue islet amyloid polypeptide (IAPP), are observed near pancreatic beta-cells of type II diabetics, with their presence strongly correlating with a loss of beta-cell mass and decreased pancreatic function. Although beta-cell membranes have been implicated as the likely target of amyloidogenic IAPP toxicity, interactions between membranes and IAPP in the fibrillar state have not been well characterized. In this study, turbidity measurements were conducted to provide a detailed description of the binding reaction between IAPP fibrils and lipid vesicles made from phosphatidylcholine. Kinetic data representing the rate and extent of the fibril-vesicle binding reaction were well described by an empirical double-exponential equation. The extent of binding was found to increase with increasing amyloid fibril concentration. Modification of the vesicle composition significantly altered the observed binding reaction kinetics, with the change quantified using the parameters obtained from the double exponential fitting analysis. When the vesicles contained a significant amount of the lipid, phosphatidylglycerol, substantial sedimentation of the vesicles under gravity was detected following the initial binding reaction. To rationalize the observed kinetic binding data, a mesoscopic simulation model was developed based on a hard particle representation of the species involved. In light of the observed data and simulation predictions the potential roles of IAPP amyloid fibrils in membrane binding are discussed.

One of the most fundamental questions in control of gene expression in mammals is how epigenetic methylation patterns of DNA and histones are established, erased, and recognized. This central process in controlling metazoan gene expression includes coordinated covalent modifications of DNA and its associated histones. This article focuses on recent developments in characterizing the functional links between methylation status of the DNA and of two particularly important histone marks. Mammalian DNA methylation is intricately connected to the presence of unmodified lysine 4 and methylated lysine 9 residues in histone H3. An interconnected network of methyltransferases, demethylases, and accessory proteins is responsible for changing or maintaining the modification status of specific regions of chromatin. The structural and functional interactions among members of this network are critical to processes that include imprinting and differentiation, and dysregulation of which is associated with disorders ranging from inflammation to cancer.

In Synechocystis sp. PCC 6803, PsbQ is associated with photosystem II (PSII) complexes with highest activity and stability. However, this subunit is not found in PSII structures from Thermosynechococcus elongatus. We present the first crystal structure of cyanobacterial PsbQ solved in the presence and absence of Zn2+. The three-dimensional structure has a well-defined helical core, composed of four helices arranged in an up-down-up-down fold. A conserved potential interaction site composed of a divalent metal binding site and adjacent hydrophobic pocket has been identified.

Various mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to susceptibility for both familial and idiopathic late-onset Parkinson's disease (PD). In this study, we have demonstrated that phosphorylation of MBP and LRRKtide by the LRRK2 G2019S mutant was activated by Mn²⁺ <i>in vitro</i>. This enhanced G2019S kinase activity was due to the combination of an increase in kinase and a decrease in ATPase activity by Mn²⁺. Compared to 10 mM Mg²⁺, 1 mM Mn²⁺ reduced ATP </i>K_m</i> for G2019S from 103 to 1.8 microM and only modestly reduced <i>k_cat</i> (2.5-fold); as a result, the Mn²⁺ increased its <i>k_cat/K_m</i> by 22-fold. This change in ATP <i>K_m</i> was due in large part to an increase in nucleotide affinity. While Mn²⁺ also increased ATP affinity and had similar effects on <i>k_cat/K_m</i> for LRRK2 WT and R1441C enzymes, it reduced their <i>k_cat</i> values significantly by 13- to 17-fold. Consequently, the difference in the kinase activity between G2019S and other LRRK2 variants was enhanced from about 2-fold in Mg²⁺ to 10-fold in Mn²⁺ at saturating ATP concentrations relative to its <i>K_m</i>. Furthermore, while Mg²⁺ yielded optimal <i>V_max</i> values at Mg²⁺ concentration greater than 5 mM, the optimal Mn²⁺ concentrations for activating LRRK2 catalysis was in the micromolar range with increasing Mn²⁺ above 1 mM causing a decrease in enzyme activity. Finally, despite the large but expected differences in IC₅₀ tested at 100 microM ATP, the K_i values of a small set of LRRK2 ATP-competitive inhibitors were within 5-fold between Mg²⁺- and Mn²⁺-mediated reactions except AMP-CPP, an ATP analog.

Long repeated sequences of DNA and their associated secondary structure govern the development and severity of a significant class of neurological diseases. Utilizing the effect of base stacking on fluorescence quantum yield, 2-aminopurine substitutions for adenine previously demonstrated sequestered bases in the stem and exposed bases in the loop for an isolated (CAG)₈ sequence. The present studies evaluate (CAG)₈ that is incorporated into a duplex, as this three-way junction is a relevant model for intermediates that lead to repeat expansion during DNA replication and repair. From an energetic perspective, thermally-induced denaturation indicates that the duplex arms dictate stability and that secondary structure of the repeated sequence is disrupted. Substitutions with 2-aminopurine probe base exposure throughout this structure, and two conclusions about secondary structure are derived. First, the central region of (CAG)₈ is more solvent-exposed than single-stranded DNA, which suggests that hairpin formation in the repeated sequence is disrupted. Second, base stacking becomes compromised in the transition from duplex to (CAG)₈, resulting in bases that are most similar to single-stranded DNA at the junction. Thus, an open (CAG)₈ loop and exposed bases in the arms indicate that the strand junction has a profound influence repeated sequences within three-way junctions.

D, L-Sulforaphane (SFN), a synthetic analogue of broccoli-derived L-isomer, is a highly promising cancer chemopreventive agent substantiated by inhibition of chemically-induced cancer in rodents and prevention of cancer development and distant site metastasis in transgenic mouse models of cancer. SFN is also known to inhibit growth of human cancer cells in association with cell cycle arrest and reactive oxygen species-dependent apoptosis, but the mechanism of these cellular responses to SFN exposure is not fully understood. Because 4-hydroxynonenal (4-HNE), a product of lipid peroxidation (LPO), the formation of which is regulated by hGSTA1-1, assumes a pivotal role in oxidative stress-induced signal transduction, the present study investigated its contribution in growth arrest and apoptosis induction by SFN using HL60 and K562 human leukemic cell lines as a model. The SFN-induced formation of 4-HNE was suppressed in hGSTA1-1 over expressing cells, which also acquired resistance to SFN-induced cytotoxicity, cell cycle arrest, and apoptosis. While resistance to SFN-induced cell cycle arrest by ectopic expression of hGSTA1-1 was associated with changes in levels of G2/M regulatory proteins, resistance to apoptosis correlated with increased Bcl-xL/Bax ratio, inhibition of nuclear translocation of AIF, and attenuated cytochrome c release in cytosol. The hGSTA1-1 over expressing cells showed enhanced cytoplasmic export of Daxx, nuclear accumulation of transcription factors Nrf2 and HSF1, and up regulation of their respective client proteins, gamma-GCS and HSP70. These findings not only reveal a central role of 4-HNE in cellular responses to SFN but also reaffirm that 4-HNE contributes to oxidative stress mediated signaling.

The FapR protein of Bacillus subtilis has been shown to play an important role in membrane lipid homeostasis. FapR acts as a repressor of many genes involved in fatty acid and phospholipid metabolism (the fap regulon). FapR binding to DNA is antagonized by malonyl-CoA, and thus FapR acts as a sensor of the status of fatty acid biosynthesis. However, malonyl-CoA is utilized for fatty acid synthesis only following its conversion to malonyl-ACP, which plays a central role in the initiation and elongation cycles carried out by the type II fatty acid synthase. Using in vitro transcription studies and isothermal titration calorimetry, we show here that malonyl-ACP binds FapR disrupting the repressor-operator complex with a similar affinity to that of its precursor malonyl-CoA. NMR experiments reveal that there is not any protein-protein recognition between ACP and FapR. These findings are consistent with the crystal structure of malonyl-ACP, which shows that the malonyl-phosphopantetheine moiety protrudes away from the protein core and thus can act as an effector ligand. Therefore, FapR regulates the expression of the fap regulon in response to the composition of the malonyl-phosphopantetheine pool. This mechanism ensures that fatty acid biosynthesis in B. subtilis is finely regulated at the transcriptional level by sensing the concentrations of the two first intermediates (malonyl-CoA and malonyl-ACP), in order to balance the production of membrane phospholipids.

The extracellular senile plaques prevalent in brain tissue in Alzheimer's disease (AD) are composed of amyloid fibrils formed by the Abeta peptide. These fibrils have been traditionally believed to feature in neurotoxicity; however, numerous recent studies provide evidence that cytotoxicity in AD may be associated with low molecular weight oligomers of Abeta that associate with neuronal membranes and may lead to membrane permeabilization and disruption of the ion balance in the cell. The underlying mechanism leading to disruption of the membrane is the subject of many recent studies. Here we report the application of single molecule optical detection, using fluorescently labeled human Alphabeta40, combined with membrane conductivity measurements, to monitor the interaction of single oligomeric peptide structures with model planar black lipid membranes (BLM). In a qualitative study, we show that the binding of Alphabeta to the membrane can be described by three distinctly different behaviors, depending on the Alphabeta monomer concentration. For concentrations much below 10 nM, there is uniform binding of monomers over the surface of the membrane with no evidence of oligomer formation or membrane permeabilization. Between 10 nM and a few 100 nM, the uniform monomer binding is accompanied by the presence of peptide species ranging from dimers to small oligomers. The dimers are not found to permeabilize the membrane but the larger oligomers lead to permeabilization with individual oligomers producing ion conductances of less than 10 pS/pore. At higher concentration, perhaps beyond physiologically relevant concentrations, larger extended and dynamic structures are found with large conductance (100's of pS) suggesting major disruption of the membrane.

Transcription by all RNA polymerases (RNAP) requires a series of large-scale conformational changes to form the transcriptionally-competent open complex RPo. At the lambdaPR promoter, E. coli sigma70 RNAP first forms a wrapped, closed 100 bp complex I1. The subsequent step opens the entire 13 base DNA bubble, creating the relatively unstable (open) complex I2. Additional conformational changes convert I2 to the stable RPo. Here we probe these events by dissecting the effects of Na+ salts of Glu-, F- and Cl- on each step in this critical process. Rapid mixing and nitrocellulose filter binding reveal that the binding constant for I1 at 25 oC is ~30-fold larger in Glu- than in Cl- at the same [Na+], with the same log-log [salt] dependence for both anions. In contrast, both the rate constant and equilibrium constant for DNA opening (I1 to I2) are only weakly [salt]-dependent, and the opening rate constant is insensitive to replacement of Cl- by Glu-. These very small effects of [salt] on a process (DNA opening) which is strongly [salt]-dependent in solution may indicate that the backbones of both DNA strands interact with polymerase throughout the process, and/or that compensation is present between ion uptake and release. Replacement of Cl- by Glu- or F- at 25 oC greatly increases the lifetime of RPo and greatly reduces its [salt]-dependence. By analogy to Hofmeister salt effects on protein folding, we propose that the excluded anions Glu- and F- drive the folding and assembly of the RNAP clamp/jaw domains in the conversion of I2 to RPo, while Cl- does not. Because the Hofmeister effect of these anions largely compensates for the destabilizing coulombic effect of any salt on the binding of this assembly to downstream promoter DNA, RPo remains long-lived even at 0.5 M Na+ in Glu- or F- salts. The observation that Esigma70 RPo are exceedingly long-lived in moderate to high [Glu-] argues that Esigma70 RNAP do not dissociate from strong promoters in vivo when the cytoplamsic increases during osmotic stress.

The structure of the 1,N(2)-ethenodeoxyguanosine lesion (1,N(2)-epsilondG) has been characterized in 5'-d(CGCATXGAATCC)-3'.5'-d(GGATTCATGCG)-3' (X = 1,N(2)-epsilondG), in which there is no dC opposite the lesion. This duplex (named the 1-BD duplex) models the product of translesion bypass of 1,N(2)-epsilondG by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) [Zang, H., Goodenough, A. K., Choi, J. Y., Irimia, A., Loukachevitch, L. V., Kozekov, I. D., Angel, K. C., Rizzo, C. J., Egli, M., and Guengerich, F. P. (2005) J. Biol. Chem. 280, 29750-29764], leading to a one-base deletion. The T(m) of this duplex is 6 degrees C higher than that of the duplex in which dC is present opposite the 1,N(2)-epsilondG lesion and 8 degrees C higher than that of the unmodified 1-BD duplex. Analysis of NOEs between the 1,N(2)-epsilondG imidazole and deoxyribose H1' protons and between the 1,N(2)-epsilondG etheno H6 and H7 protons and DNA protons establishes that 1,N(2)-epsilondG adopts the anti conformation about the glycosyl bond and that the etheno moiety is accommodated within the helix. The resonances of the 1,N(2)-epsilondG H6 and H7 etheno protons shift upfield relative to the monomer 1,N(2)-epsilondG, attributed to ring current shielding, consistent with their intrahelical location. NMR data reveal that Watson-Crick base pairing is maintained at both the 5' and 3' neighbor base pairs. The structure of the 1-BD duplex has been refined using molecular dynamics calculations restrained by NMR-derived distance and dihedral angle restraints. The increased stability of the 1,N(2)-epsilondG lesion in the absence of the complementary dC correlates with the one-base deletion extension product observed during the bypass of the 1,N(2)-epsilondG lesion by the Dpo4 polymerase, suggesting that stabilization of this bulged intermediate may be significant with regard to the biological processing of the lesion.

The membrane protein, Rv0899 (OmpATb), from M. tuberculosis, has been proposed to act as an outer membrane porin and to contribute to the bacterium's adaptation to the acidic environment of the phagosome during infection. The gene is restricted to pathogenic mycobacteria and, thus, is an attractive candidate for the development of anti-TB chemotherapy. The 326-residue protein contains three domains: an N-terminal domain (residues 1-72) which includes a sequence of 20 hydrophobic amino acids that is required for membrane translocation; a central B domain (residues 73-200) with homology to the conserved putative lipid-binding BON (bacterial OsmY and nodulation) superfamily; and a C domain (residues 201-326) with homology to the OmpA-C-like superfamily of periplasmic peptidoglycan-binding sequences, found in several types of bacterial membrane proteins, including in the C-terminus of the E. coli outer membrane protein OmpA. We have characterized the structure and dynamics of the B and C domains, and have determined the three-dimensional structure of the B domain. Rv0899 does not form a transmembrane beta-barrel. Residues 73 to 326 form a mixed alpha/beta globular structure, encompassing two independently folded modules corresponding to the B and C domains connected by a flexible linker. The B domain folds with three parallel/antiparallel alpha-helices packed against six parallel/antiparallel beta-strands which form a flat beta-sheet. The core is hydrophobic while the exterior is polar and predominantly acidic. The structure of a BON homology domain is revealed here for the first time. In light of this unexpected structure, it is hard to reconcile an outer membrane porin activity with the central domain of the protein. The structure of the B domain and the overall architecture of the protein suggest alternative modes of membrane association.

Identification of diseases caused by protein misfolding has increased interest in the way proteins adopt non-native conformations and form aggregates. In this study we address the question of how proteins sharing the same fold respond to destabilising environmental conditions. We have studied the behaviour of two members of the cystatin superfamily, MNEI, a single chain monellin and orizacystatin_I, a plant cystatin. Despite the close similarity of their three dimensional architecture, these two proteins aggregate in a different way: MNEI gives rise to amyloid aggregation whereas orizacystatin_I yields amorphous aggregates. Mutants of orizacystatin_I, designed to make it more similar to MNEI, generally behave like the parent protein, but a construct devoid of the disordered N- and C-terminal sequences does show a tendency to form amyloid fibres. Our results suggest that precise sequence details may be more important than the three-dimensional architecture in determining the type of aggregate formed. Orizacystatin_I appears to be a very promising model system for further studies of protein aggregation.

Glutamate receptors are important potential drug targets for cognitive enhancement and the treatment of schizophrenia in part because they are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system. One approach to the application of therapeutic agents to the AMPA subtype of glutamate receptors is the use of allosteric modulators, which promote dimerization by binding to a dimer interface thereby reducing desensitization and deactivation. AMPA receptors exist in two alternatively spliced variants (flip and flop) that differ in desensitization and receptor activation profiles. Most of the structural information on modulators of the AMPA receptor target the flip subtype. We report here the crystal structure of the flop-selective allosteric modulator, PEPA, bound to the binding domains of the GluA2 and GluA3 flop isoforms of AMPA receptors. Specific hydrogen bonding patterns can explain the preference for the flop isoform. This includes a bidentate hydrogen bonding pattern between PEPA and N754 of the flop isoforms of GluA2 and GluA3 (the corresponding position in the flip isoform is S754). Comparison with other allosteric modulators provides a framework for the development of new allosteric modulators with preferences for either the flip or flop isoforms. In addition to interactions with N/S754, specific interactions of the sulfonamide with conserved residues in the binding site are characteristics of a number of allosteric modulators. These, in combination, with variable interactions with five subsites on the binding surface lead to different stoichiometries, orientations within the binding pockets, and functional outcomes.