Biomedical subjects

John P Marino

Publications and source records attributed to John P Marino.

14 recordsLinked to original sources

PubMed2006

The receptor-bound "empty pocket" state of the heterotrimeric G-protein alpha-subunit is conformationally dynamic.

Heterotrimeric G-protein activation by a G-protein-coupled receptor (GPCR) requires the propagation of structural signals from the receptor-interacting surfaces to the guanine nucleotide-binding pocket. To probe conformational changes in the G-protein alpha-subunit (G(alpha)) associated with activated GPCR (R*) interactions and guanine nucleotide exchange, high-resolution solution NMR methods are being applied in studying signaling of the G-protein, transducin, by light-activated rhodopsin. Using these methods, we recently demonstrated that an isotope-labeled G(alpha) reconstituted heterotrimer forms functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin and a soluble mimic of R*, both of which trigger guanine nucleotide exchange [Ridge, K. D., et al. (2006) J. Biol. Chem. 281, 7635-7648]. Here, it is shown that both light-activated rhodopsin and the soluble mimic of R form trapped intermediate complexes with a GDP-released "empty pocket" state of the heterotrimer in the absence of GTP (or GTPgammaS). In contrast to guanine nucleotide-bound forms of G(alpha), the NMR spectra of the GDP-released, R-bound empty pocket state of G(alpha) display severe line broadening suggestive of a dynamic intermediate state. Interestingly, the conformation of a GDP-depleted, Mg(2+)-bound state of G(alpha) generated in a manner independent of R* does not exhibit a similar degree of line broadening but rather appears structurally similar to the GDP/Mg(2+)-bound form of the protein. Taken together, these results suggest that R*-mediated changes in the receptor-interacting regions of G(alpha), and not the absence of bound guanine nucleotide, are the predominant factors which dictate G(alpha) conformation and dynamics in this R*-bound state of the heterotrimer.

Animals↗

PubMed2006

Conformational changes associated with receptor-stimulated guanine nucleotide exchange in a heterotrimeric G-protein alpha-subunit: NMR analysis of GTPgammaS-bound states.

Solution NMR studies of a (15)N-labeled G-protein alpha-subunit (G(alpha)) chimera ((15)N-ChiT)-reconstituted heterotrimer have shown previously that G-protein betagamma-subunit (G(betagamma)) association induces a "pre-activated" conformation that likely facilitates interaction with the agonist-activated form of a G-protein-coupled receptor (R*) and guanine nucleotide exchange (Abdulaev, N. G., Ngo, T., Zhang, C., Dinh, A., Brabazon, D. M., Ridge, K. D., and Marino, J. P. (2005) J. Biol. Chem. 280, 38071-38080). Here we demonstrated that the (15)N-ChiT-reconstituted heterotrimer can form functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin (R*), as well as a soluble mimic of R*. NMR methods were used to track R*-triggered guanine nucleotide exchange and release of guanosine 5'-O-3-thiotriphosphate (GTPgammaS)/Mg(2+)-bound ChiT. A heteronuclear single quantum correlation (HSQC) spectrum of R*-generated GTPgammaS/Mg(2+)-bound ChiT revealed (1)HN, (15)N chemical shift changes relative to GDP/Mg(2+)-bound ChiT that were similar, but not identical, to those observed for the GDP.AlF(4)(-)/Mg(2+)-bound state. Line widths observed for R*-generated GTPgammaS/Mg(2+)-bound (15)N-ChiT, however, indicated that it is more conformationally dynamic relative to the GDP/Mg(2+)- and GDP.AlF(4)(-)/Mg(2+)-bound states. The increased dynamics appeared to be correlated with G(betagamma) and R* interactions because they are not observed for GTPgammaS/Mg(2+)-bound ChiT generated independently of R*. In contrast to R*, a soluble mimic that does not catalytically interact with G-protein (Abdulaev, N. G., Ngo, T., Chen, R., Lu, Z., and Ridge, K. D. (2000) J. Biol. Chem. 275, 39354-39363) is found to form a stable complex with the GTPgammaS/Mg(2+)-exchanged heterotrimer. The HSQC spectrum of (15)N-ChiT in this complex displays a unique chemical shift pattern that nonetheless shares similarities with the heterotrimer and GTPgammaS/Mg(2+)-bound ChiT. Overall, these results demonstrated that R*-induced changes in G(alpha) can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation.

Amino Acid Sequence↗

PubMed2005

A ribose sugar conformational switch in the LTR-retrotransposon Ty3 polypurine tract-containing RNA/DNA hybrid.

To probe structural features of a polypurine tract (PPT) that mediate its specific recognition and processing, a model 20 bp RNA/DNA hybrid duplex, which includes the full PPT sequence of the Saccharomyces cerevisiae LTR-retrotransposon Ty3, has been investigated using solution NMR spectroscopy. While homonuclear NOESY and DQF-COSY analyses indicate that this PPT-containing RNA/DNA hybrid adopts an overall A-form-like helical geometry, an unexpected sugar pucker switch has been detected for the ribose at position +1, relative to the cleavage site, on the RNA strand. A model of the conformational changes induced by the A- to B-type sugar pucker switch shows a significant change in the backbone trajectory of the RNA strand, which alters the presentation of backbone phosphate and 2' hydroxyl groups 3' of this residue. This observation implies that part of the mechanism governing RNase H fidelity may be through distortion of the RNA/DNA helix one base ahead of the scissile bond.

Carbohydrate Conformation↗

PubMed2005

Examining Ty3 polypurine tract structure and function by nucleoside analog interference.

We have combined nucleoside analog interference with chemical footprinting, thermal denaturation, NMR spectroscopy, and biochemical studies to understand recognition of the polypurine tract (PPT) primer of the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3 by its cognate reverse transcriptase. Locked nucleic acid analogs, which constrain sugar ring geometry, were introduced pairwise throughout the PPT (-)-DNA template, whereas abasic tetrahydrofuran linkages, which lack the nucleobase but preserve the sugar phosphate backbone, were introduced throughout the (-)-strand DNA template and (+)-strand RNA primer. Collectively, our data suggest that both the 5'- and 3'-portions of the PPT-containing RNA/DNA hybrid are sensitive to nucleoside analog substitution, whereas the intervening region can be modified without altering cleavage specificity. These two regions most likely correspond to portions of the PPT that make close contact with the Ty3 reverse transcriptase thumb subdomain and RNase H catalytic center, respectively. Achieving a similar phenotype with nucleoside analogs that have different effects on duplex geometry reveals structural features that are important mediators of Ty3 PPT recognition. Finally, the results from introducing tetrahydrofuran lesions around the scissile PPT/unique 3'-sequence junction indicate that template nucleobase -1 is dispensable for catalysis, whereas a primer nucleobase on either side of the junction is necessary.

Base Sequence↗

PubMed2005

Heterotrimeric G-protein alpha-subunit adopts a "preactivated" conformation when associated with betagamma-subunits.

Activation of a heterotrimeric G-protein by an agonist-stimulated G-protein-coupled receptor requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide binding pocket of the G-protein. To probe the molecular basis of this signaling process, we are applying high resolution NMR to track structural changes in an isotope-labeled, full-length G-protein alpha-subunit (G(alpha)) chimera (ChiT) associated with G-protein betagamma-subunit (G(betagamma)) and activated receptor (R(*)) interactions. Here, we show that ChiT can be functionally reconstituted with G(betagamma) as assessed by aluminum fluoride-dependent changes in intrinsic tryptophan fluorescence and light-activated rhodopsin-catalyzed guanine nucleotide exchange. We further show that (15)N-ChiT can be titrated with G(betagamma) to form stable heterotrimers at NMR concentrations. To assess structural changes in ChiT upon heterotrimer formation, HSQC spectra of the (15)N-ChiT-reconstituted heterotrimer have been acquired and compared with spectra obtained for GDP/Mg(2+)-bound (15)N-ChiT in the presence and absence of aluminum fluoride and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS)/Mg(2+)-bound (15)N-ChiT. As anticipated, G(betagamma) association with (15)N-ChiT results in (1)HN, (15)N chemical shift changes relative to the GDP/Mg(2+)-bound state. Strikingly, however, most (1)HN, (15)N chemical shift changes associated with heterotrimer formation are the same as those observed upon formation of the GDP.AlF(4)(-)/Mg(2+)- and GTPgammaS/Mg(2+)-bound states. Based on these comparative analyses, assembly of the heterotrimer appears to induce structural changes in the switch II and carboxyl-terminal regions of G(alpha) ("preactivation") that may facilitate the interaction with R(*) and subsequent GDP/GTP exchange.

Escherichia coli↗

PubMed2005

Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein alpha-subunit.

Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein alpha-subunit (G(alpha)) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled G(alpha) chimera (approximately 40 kDa polypeptide) has been tested. The results show that a prodomain fused G(alpha) chimera can be expressed to levels approaching 6-8 mg/l in minimal media and that the processed, mature protein exhibits properties similar to those of G(alpha) isolated from natural sources. To assay for the functional integrity of the purified G(alpha) chimera at NMR concentrations and probe for changes in the structure and dynamics of G(alpha) that result from activation, 15N-HSQC spectra of the GDP/Mg2+ bound form of G(alpha) obtained in the absence and presence of aluminum fluoride, a well known activator of the GDP bound state, have been acquired. Comparisons of the 15N-HSQC spectra reveals a number of changes in chemical shifts of the 1HN, 15N crosspeaks that are discussed with respect to expected changes in the protein conformation associated with G(alpha) activation.

GTP-Binding Protein alpha Subunits, Gi-Go↗

PubMed2004

Ligand-induced changes in 2-aminopurine fluorescence as a probe for small molecule binding to HIV-1 TAR RNA.

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated in part through an interaction between the virally encoded trans-activator protein Tat and the trans-activator responsive region (TAR) of the viral RNA genome. Because TAR is highly conserved and its interaction with Tat is required for efficient viral replication, it has received much attention as an antiviral drug target. Here, we report a 2-aminopurine (2-AP) fluorescence-based assay for evaluating potential TAR inhibitors. Through selective incorporation of 2-AP within the bulge (C23 or U24) of a truncated form of the TAR sequence (delta TAR-ap23 and delta TAR-ap24), binding of argininamide, a 24-residue arginine-rich peptide derived from Tat, and Neomycin has been characterized using steady-state fluorescence. Binding of argininamide to the 2-AP deltaTAR constructs results in a four- to 11-fold increase in fluorescence intensity, thus providing a sensitive reporter of that interaction (KD approximately 1 mM). Similarly, binding of the Tat peptide results in an initial 14-fold increase in fluorescence (KD approximately 25 nM), but is then followed by a slight decrease that is attributed to an additional, lower-affinity association(s). Using the deltaTAR-ap23 and TAR-ap24 constructs, two classes of Neomycin binding sites are detected; the first molecule of antibiotic binds as a noncompetitive inhibitor of Tat/argininamide (KD approximately 200 nM), whereas the second, more weakly bound molecule(s) becomes associated in a presumably nonspecific manner (KD approximately 4 microM). Taken together, the results demonstrate that the 2-AP fluorescence-detected binding assays provide accurate and general methods for quantitatively assessing TAR interactions.

2-Aminopurine↗

PubMed2004

A proton-coupled dynamic conformational switch in the HIV-1 dimerization initiation site kissing complex.

In HIV type 1 (HIV-1), the dimerization initiation site (DIS) is the sequence primarily responsible for initiating the noncovalent linkage of two homologous strands of genomic RNA during viral assembly. The DIS loop contains an autocomplementary hexanucleotide sequence and forms a symmetric homodimer through a loop-loop kissing interaction. In a structural rearrangement catalyzed by the HIV-1 nucleocapsid protein (NCp7) and suggested to be associated with maturation of the budded viral particle, the DIS converts from a metastable kissing dimer to an extended duplex. Here, we demonstrate that the DIS kissing dimer displays localized conformational dynamics that result from the specific protonation of the N1 base nitrogen of the DIS loop residue A272 at near-physiological pH. The rate of NCp7-catalyzed maturation of the DIS kissing dimer is also shown to directly correlate with the observed proton-coupled conformational dynamics, where NCp7 is found to convert the dynamic A272 protonated state with a faster rate. Taken together, these results reveal a previously undescribed role for base protonation in modulating local RNA structure and demonstrate a mechanism for promoting the chaperone-mediated structural rearrangement of a kinetically trapped RNA conformational state.

Catalysis↗

PubMed2004

Homonuclear Hartmann-Hahn transfer with reduced relaxation losses by use of the MOCCA-XY16 multiple pulse sequence.

Homonuclear Hartmann-Hahn transfer is one of the most important building blocks in modern high-resolution NMR. It constitutes a very efficient transfer element for the assignment of proteins, nucleic acids, and oligosaccharides. Nevertheless, in macromolecules exceeding approximately 10 kDa TOCSY-experiments can show decreasing sensitivity due to fast transverse relaxation processes that are active during the mixing periods. In this article we propose the MOCCA-XY16 multiple pulse sequence, originally developed for efficient TOCSY transfer through residual dipolar couplings, as a homonuclear Hartmann-Hahn sequence with improved relaxation properties. A theoretical analysis of the coherence transfer via scalar couplings and its relaxation behavior as well as experimental transfer curves for MOCCA-XY16 relative to the well-characterized DIPSI-2 multiple pulse sequence are given.

Algorithms↗

PubMed2003

Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1.

Rev is an essential regulatory HIV-1 protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome, activating the switch between viral latency and active viral replication. Previously, we have shown that selective incorporation of the fluorescent probe 2-aminopurine (2-AP) into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of an arginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescence methods. Using these fluorescence and nuclear magnetic resonance (NMR) methods, proflavine has been identified, through a limited screen of selected small heterocyclic compounds, as a specific and high-affinity RRE-IIB binder which inhibits the interaction of the Rev peptide with RRE-IIB. Direct and competitive 2-AP fluorescence binding assays reveal that there are at least two classes of proflavine binding sites on RRE-IIB: a high-affinity site that competes with the Rev peptide for binding to RRE-IIB (K(D) approximately 0.1 +/- 0.05 microM) and a weaker binding site(s) (K(D) approximately 1.1 +/- 0.05 microM). Titrations of RRE-IIB with proflavine, monitored using (1)H NMR, demonstrate that the high-affinity proflavine binding interaction occurs with a 2:1 (proflavine:RRE-IIB) stoichiometry, and NOEs observed in the NOESY spectrum of the 2:1 proflavine.RRE-IIB complex indicate that the two proflavine molecules bind specifically and close to each other within a single binding site. NOESY data further indicate that formation of the 2:1 proflavine.RRE-IIB complex stabilizes base pairing and stacking within the internal purine-rich bulge of RRE-IIB in a manner analogous to what has been observed in the Rev peptide.RRE-IIB complex. The observation that proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinity site opens the possibility for rational drug design based on linking and modifying it and related compounds.

2-Aminopurine↗

PubMed2003

JE-TROSY: combined J- and TROSY-spectroscopy for the measurement of one-bond couplings in macromolecules.

With the application of RDCs in high-resolution NMR studies of macromolecules, there has been an interest in the development of accurate, sensitive methods for measuring 15N-1H and 13C-1H one-bond coupling constants. Most methods for determining these couplings are based on the measurement of the displacement between cross-peak components in J-coupled spectra. However, for large macromolecules and macromolecular complexes, these methods are often unreliable since differential relaxation can significantly broaden one of the multiplet components (i.e., the anti-TROSY component) and thereby make accurate determination of its position difficult. To overcome this problem, a J-evolved transverse relaxation optimized (JE-TROSY) method is presented for the determination of one-bond couplings that involves J-evolution of the sharpest cross-peak multiplet component selected in a TROSY experiment. Couplings are measured from the displacement of the TROSY component in the additional J-evolution dimension relative to a zero frequency origin. The JE-TROSY method is demonstrated on uniformly labeled 15N, 13C-labeled RNA and peptide samples, as well as with an RNA-protein complex, in which the protein is uniformly 15N, 13C-labeled. In all cases, resolved, sensitive spectra are obtained from which heteronuclear one-bond J-couplings could be accurately and easily measured.

Algorithms↗

PubMed2002

Mechanism of nucleocapsid protein catalyzed structural isomerization of the dimerization initiation site of HIV-1.

Dimerization of two homologous strands of genomic RNA is an essential feature of retroviral replication. In the human immunodeficiency virus type 1 (HIV-1), a conserved stem-loop sequence, the dimerization initiation site (DIS), has been identified as the domain primarily responsible for initiation of this aspect of viral assembly. The DIS loop contains an autocomplementary hexanucleotide sequence flanked by highly conserved 5' and 3' purines and can form a homodimer through a loop-loop kissing interaction. In a structural rearrangement activated by the HIV-1 nucleocapsid protein (NCp7) and considered to be associated with viral particle maturation, the DIS dimer converts from an intermediate kissing to an extended duplex isoform. Using 2-aminopurine (2-AP) labeled sequences derived from the DIS(Mal) variant and fluorescence methods, the two DIS dimer isoforms have been unambiguously distinguished, allowing a detailed examination of the kinetics of this RNA structural isomerization and a characterization of the role of NCp7 in the reaction. In the presence of divalent cations, the DIS kissing dimer is found to be kinetically trapped and converts to the extended duplex isoform only upon addition of NCp7. NCp7 is demonstrated to act catalytically in inducing the structural isomerization by accelerating the rate of strand exchange between the two hairpin stem helices, without disruption of the loop-loop helix. Observation of an apparent maximum conversion rate for NCp7-activated DIS isomerization, however, requires protein concentrations in excess of the 2:1 stoichiometry estimated for high-affinity NCp7 binding to the DIS kissing dimer, indicating that transient interactions with additional NCp7(s) may be required for catalysis.

2-Aminopurine↗

PubMed2002

Observation of H-bond mediated 3hJH2H3 coupling constants across Watson-Crick AU base pairs in RNA.

3hJH2H3 trans-hydrogen bond scalar coupling constants have been observed for the first time in Watson-Crick AU base pairs in uniformly 15N-labeled RNA oligonucleotides using a new 2hJNN-HNN-E. COSY experiment. The experiment utilizes adenosine H2 (AH2) for original polarization and detection, while employing 2hJNN couplings for coherence transfer across the hydrogen bonds (H-bonds). The H3 protons of uracil bases are unperturbed throughout the experiment so that these protons appear as passive spins in E. COSY patterns. 3hJH2H3 coupling constants can therefore be accurately measured in the acquisition dimension from the displacement of the E. COSY multiplet components, which are separated by the relatively large 1JH3N3 coupling constants in the indirect dimension of the two-dimensional experiment. The 3hJH2H3 scalar coupling constants determined for AU base pairs in the two RNA hairpins examined here have been found to be positive and range in magnitude up to 1.8 Hz. Using a molecular fragment representation of an AU base pair, density functional theory/finite field perturbation theory (DFT/FPT) methods have been applied to attempt to predict the relative contributions of H-bond length and angular geometry to the magnitude of 3hJH2H3 coupling constants. Although the DFT/FPT calculations did not reproduce the full range of magnitude observed experimentally for the 3hJH2H3 coupling constants, the calculations do predict the correct sign and general trends in variation in size of these coupling constants. The calculations suggest that the magnitude of the coupling constants depends largely on H-bond length, but can also vary with differences in base pair geometry. The dependency of the 3hJH2H3 coupling constant on H-bond strength and geometry makes it a new probe for defining base pairs in NMR studies of nucleic acids.

Adenine↗

PubMed2002

RNA and RNA-protein complexes as targets for therapeutic intervention.

Today, the majority of pharmaceuticals developed to treat cancers and viral/bacterial infections target cellular, bacterial or viral proteins known to be associated with a given pathology. Although proteins are the focus of most current drug discovery efforts, exciting new research has recently begun which aims to exploit ribonucleic acid (RNA) and RNP particles as novel targets for pharmaceutical development. These RNA-targeted research efforts have been fueled by an increased appreciation for the central role played by RNA and RNA-protein interactions in many biological processes and diseases, together with a better understanding of RNA structure and an improvement in biophysical/biochemical techniques available to study RNA. As for protein targets, genome sequencing is greatly accelerating the identification of human and microbial RNA transcripts for targeted drug discovery. With this explosion in the number of potential RNA and RNP targets, the effective development of specific small molecule RNA-based drugs requires robust and general approaches for detecting and quantifying RNA-ligand interactions, which can be used as high-throughput screens (HTS) and for obtaining rapid structural information to guide rational drug design. In this review, an overview of the potential for therapeutic intervention based on RNA and RNP targets is presented, together with recent efforts to develop generally useful nuclear magnetic resonance (NMR) and fluorescence binding assays for screening and optimizing drugs aimed at RNA and RNP targets.

Animals↗