Excited states of ribosome translocation revealed through integrative molecular modeling.
Ontology highlight
ABSTRACT:
SUBMITTER: Whitford PC
PROVIDER: S-EPMC3223460 | biostudies-other | 2011 Nov
REPOSITORIES: biostudies-other
Publications
Excited states of ribosome translocation revealed through integrative molecular modeling.
Whitford Paul C PC Ahmed Aqeel A Yu Yanan Y Hennelly Scott P SP Tama Florence F Spahn Christian M T CM Onuchic José N JN Sanbonmatsu Karissa Y KY
Proceedings of the National Academy of Sciences of the United States of America 20111111 47
The dynamic nature of biomolecules leads to significant challenges when characterizing the structural properties associated with function. While X-ray crystallography and imaging techniques (such as cryo-electron microscopy) can reveal the structural details of stable molecular complexes, strategies must be developed to characterize configurations that exhibit only marginal stability (such as intermediates) or configurations that do not correspond to minima on the energy landscape (such as trans ...[more]
Similar Datasets
Project description:Localized protein synthesis is a fundamental mechanism for creating distinct subcellular environments. Here we developed a generalizable proximity-specific ribosome profiling strategy that enables global analysis of translation in defined subcellular locations. We applied this approach to the endoplasmic reticulum (ER) in yeast and mammals. We observed the large majority of secretory proteins to be cotranslationally translocated, including substrates capable of posttranslational insertion in vitro. Distinct translocon complexes engaged nascent chains at different points during synthesis. Whereas most proteins engaged the ER immediately after or even before signal sequence (SS) emergence, a class of Sec66-dependent proteins entered with a looped SS conformation. Finally, we observed rapid ribosome exchange into the cytosol after translation termination. These data provide insights into how distinct translocation mechanisms act in concert to promote efficient cotranslational recruitment.
| S-EPMC4285348 | biostudies-literature
Project description:A multistate energy decomposition analysis (MS-EDA) method is described to dissect the energy components in molecular complexes in excited states. In MS-EDA, the total binding energy of an excimer or an exciplex is partitioned into a ground-state term, called local interaction energy, and excited-state contributions that include exciton excitation energy, superexchange stabilization, and orbital and configuration-state delocalization. An important feature of MS-EDA is that key intermediate states associated with different energy terms can be variationally optimized, providing quantitative insights into widely used physical concepts such as exciton delocalization and superexchange charge-transfer effects in excited states. By introducing structure-weighted adiabatic excitation energy as the minimum photoexcitation energy needed to produce an excited-state complex, the binding energy of an exciplex and excimer can be defined. On the basis of the nature of intermolecular forces through MS-EDA analysis, it was found that molecular complexes in the excited states can be classified into three main categories, including (1) encounter excited-state complex, (2) charge-transfer exciplex, and (3) intimate excimer or exciplex. The illustrative examples in this Perspective highlight the interplay of local excitation polarization, exciton resonance, and superexchange effects in molecular excited states. It is hoped that MS-EDA can be a useful tool for understanding photochemical and photobiological processes.
| S-EPMC10369419 | biostudies-literature
Project description:Exchange proteins directly activated by cAMP (EPAC1 and EPAC2) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we performed detailed Small-Angle X-ray Scattering (SAXS) analysis of EPAC1 in its apo (inactive), cAMP-bound, and effector (Rap1b)-bound states. Our study demonstrates that we can model the solution structures of EPAC1 in each state using ensemble analysis and homology models derived from the crystal structures of EPAC2. The N-terminal domain of EPAC1, which is not conserved between EPAC1 and EPAC2, appears folded and interacts specifically with another component of EPAC1 in each state. The apo-EPAC1 state is a dynamic mixture of a compact (Rg = 32.9 Å, 86%) and a more extended (Rg = 38.5 Å, 13%) conformation. The cAMP-bound form of EPAC1 in the absence of Rap1 forms a dimer in solution; but its molecular structure is still compatible with the active EPAC1 conformation of the ternary complex model with cAMP and Rap1. Herein, we show that SAXS can elucidate the conformational states of EPAC1 activation as it proceeds from the compact, inactive apo conformation through a previously unknown intermediate-state, to the extended cAMP-bound form, and then binds to its effector (Rap1b) in a ternary complex.
| S-EPMC7016869 | biostudies-literature
Project description:Ultrafast reactions activated by light absorption are governed by multidimensional excited-state (ES) potential energy surfaces (PESs), which describe how the molecular potential varies with the nuclear coordinates. ES PESs ad-hoc displaced with respect to the ground state can drive subtle structural rearrangements, accompanying molecular biological activity and regulating physical/chemical properties. Such displacements are encoded in the Franck-Condon overlap integrals, which in turn determine the resonant Raman response. Conventional spectroscopic approaches only access their absolute value, and hence cannot determine the sense of ES displacements. Here, we introduce a two-color broadband impulsive Raman experimental scheme, to directly measure complex Raman excitation profiles along desired normal modes. The key to achieve this task is in the signal linear dependence on the Frank-Condon overlaps, brought about by non-degenerate resonant probe and off-resonant pump pulses, which ultimately enables time-domain sensitivity to the phase of the stimulated vibrational coherences. Our results provide the tool to determine the magnitude and the sensed direction of ES displacements, unambiguously relating them to the ground state eigenvectors reference frame.
| S-EPMC9755279 | biostudies-literature
Project description:mRNA-tRNA translocation is a central and highly regulated process during translational elongation. Along with the mRNA, tRNA moves through the ribosome in a stepwise fashion. Using cryoelectron microscopy on ribosomes with a P-loop mutation, we have identified novel structural intermediates likely to exist transiently during translocation. Our observations suggest a mechanism by which the rate of translocation can be regulated.
| S-EPMC3064370 | biostudies-literature
Project description:Translocation of messenger and transfer RNA (mRNA and tRNA) through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. The crystal structures of three Thermus ribosome-tRNA-mRNA-EF-G complexes trapped with β,γ-imidoguanosine 5'-triphosphate (GDPNP) or fusidic acid reveal conformational changes occurring during intermediate states of translocation, including large-scale rotation of the 30S subunit head and body. In all complexes, the tRNA acceptor ends occupy the 50S subunit E site, while their anticodon stem loops move with the head of the 30S subunit to positions between the P and E sites, forming chimeric intermediate states. Two universally conserved bases of 16S ribosomal RNA that intercalate between bases of the mRNA may act as "pawls" of a translocational ratchet. These findings provide new insights into the molecular mechanism of ribosomal translocation.
| S-EPMC3979973 | biostudies-literature
Project description:Using laser capture microdissection to isolate over 100 specific cell populations, we report the profiling of prostate cancer progression from benign epithelium to metastatic disease. By analyzing these expression signatures in the context of over 15,000 â??molecular conceptsâ??, or sets of biologically connected genes, we generated an integrative model of prostate cancer progression. Keywords: disease state analysis Experiment Design: â?¢ Type of experiment: This study used microarray experiments to profile prostate cancer progression through the isolation of pure cell populations using laser capture microdissection (LCM) and OmniPlex Whole Transcriptome (WTA) Amplification. â?¢ Experimental factors: This study profiled various pure cell populations isolated by LCM from prostate tissue. â?¢ The number of hybridizations performed in the experiment: A total of 104 hybridizations were performed. â?¢ The type of reference used for the hybridizations: A single commercially available pool of benign prostate tissue (CPP, Clontech) was used as the reference in all hybridizations. For the reference sample, 10 ng of CPP total RNA was converted into a WTA cDNA library in a volume of 25 µl and five 5 µl aliquots were amplified by WTA PCR and purified. Five ng aliquots of the pooled products were subjected to a second WTA PCR amplification in the presence of amino-allyl dUTP and all products were pooled before labeling to ensure equal representation across all of the hybridizations. â?¢ Hybridization design: For all hybridizations, the experimental prostate sample isolated by LCM was labeled with Cy5 and the common reference of benign pooled prostate as described above was labeled with Cy3. â?¢ Quality control steps taken: In order to assess biological and technical reproducibility, we captured identical regions from two specimens (PCA_11 and MET_HR_3) and subjected one sample, PCA_30, to two hybridizations. For PCA_11, adjacent regions of the same section were captured and treated as separate samples. For MET_HR_3, two separate foci of the liver metastasis (from separate frozen blocks) were captured and treated as separate samples. For PCA_30, two aliquots of the primary WTA PCR products were separately subjected to a second round of WTA PCR amplification. Replicates from PCA_30 and MET_HR_3 were also hybridized to arrays from each of the print runs utilized in this study. Samples used, extract preparation and labeling: â?¢ The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: All samples used were of human origin. The table in the MIAME Checklist (Supplmentary Methods) summarizes the samples used. Hybridizations are named according to the class of sample profiled and the background color is the same as shown in Figure S3. Two print runs were used during the course of the study and the indicated run for each hybridization is given. For prostate cancer samples, the Gleason patterns present on the sample section used are indicated, as well as the Gleason patterns of the actual cells isolated by LCM. A unique case number for all profiled samples is given. Descriptions of samples are given where applicable, including the location of metastatic foci. â?¢ Manipulation of biological samples and protocols used: Laser Capture Microdissection (LCM) was performed from frozen tissue sections with the SL Microtest device using µCUT software (MMI). Approximately 10,000 cells were captured for each sample. Serial sections were used if cells could not be obtained from a single section. Total RNA was isolated from captured cells with the RNAqueous Micro kit (Ambion) and treated with DNAse I according to the manufacturer's instructions. RNA quantification was perfomed using Ribogreen (Molecular Probes). â?¢ Protocol for preparing the hybridization extract: For each sample, total RNA (~10ng) in a volume of 25 µl was converted into an OmniPlex WTA cDNA library and amplified by WTA PCR using reagents and protocols according to the beta-commercial TransPlex WTA kit (Rubicon Genomics, Ann Arbor, MI). For each sample, a single 10 µl aliquot of the WTA cDNA library was amplified by WTA PCR and products were purified. Four or five 5 ng aliquots of product were subjected to a second WTA PCR amplification in the presence of amino-allyl dUTP for post amplification labeling and products were pooled before proceeding with the hybridization. Yields after all WTA PCR amplifications were between 2 to 5 μg per reaction, and reaction progress was monitored in real time using SYBR green I (Molecular Probes) on the iCycler IQ (BioRad) or ABI 7300 Real Time PCR system (Applied Biosystems). Real-time PCR amplifications were terminated at or before plateau phase as measured by fluorescence incorporation to preserve maximum representation. â?¢ Labeling protocol(s): For all WTA amplified cDNA samples with amino-allyl dUTP incorporated (10-20 ï?g), nuclease free water was added to 500 ï?l, the sample was transferred to a Microcon YM-30 filter, and centrifuged at 13,000 rcf at RT for 12 minutes. The spin column was inverted in a new collection tube and centrifuged at 13,000 rcf for 2 min at RT. The amount of sample was measured and the volume was adjusted to 5 ï?l with nuclease free water. For labeling, 5 ï?l of 1 M sodium bicarbonate (pH 9.0) was added and allowed to incubate at RT for 15 min. Nine ï?l of anhydrous DMSO were added to Mono Reactive Cy3 and Cy5 dye packs (Amersham, Buckinghamshire, England), mixed thoroughly, and 1 ï?l of the proper dye was added to each sample. The labeling mixture was incubated in the dark at RT for 1 hour. The reaction was stopped by the addition of 9 ï?l of 4 M Hydroxylamine for 15 min. For each labeling mixture, RNase free water was added to 100 ï?l, 500 ï?l of PB buffer was added and Cy3 and Cy5 mixtures were added to separate Qiaquick (Qiagen, Valencia, CA) spin columns and centrifuged at 13,000 rcf. Columns were washed twice with 700 ï?l of buffer PE, and centrifuged dry at 13,000 rcf for 2 min. To elute, 60 ï?l of EB buffer was placed on the column, incubated for 5 min and centrifuged at 13,000 rcf for 1 min. The Cy3 labeled and Cy5 labeled cDNA for each hybridization were mixed and 1.5 ï?l were used for analysis on a ND-1000 spectrophotometer. â?¢ External controls (spikes): Not utilized Hybridization procedures and parameters: â?¢ The protocol and conditions used during hybridization, blocking and washing: For hybridization, 40 ï?g of human Cot-1 DNA (Invitrogen, Carlsbad, CA) was added to 120 ï?l of labeled cDNA, the probe was transferred to a Microcon YM-30 filter and centrifuged at 13,000 rcf for 6 min at RT. The spin column was inverted in a new collection tube and centrifuged at 13,000 rcf for 2 min at RT. The eluted probe volume was adjusted to 18.6 ï?l with nuclease free water and the following were added: 4 ï?l of yeast tRNA (10 ï?g/ï?l, Invitrogen, Carlsbad, CA), 4.9 ï?l of 20X SSC and 0.84 ï?l of 10% SDS. The probe was denatured at 100 Co for 3 min and centrifuged at 13,000 rcf for 45 sec. The probe was added directly to the microarray and a cover slip was added. Slides were hybridized overnight in DIE-TECH 1 or 5 slide hybridization chambers in a 65 Co water bath. After hybridization, cover slips were removed by incubation in 2x SSC / 0.05% SDS. Slides were then washed in 2x SSC / 0.05% SDS for 5 min and 0.2x SSC / 0.05% SDS for five minutes. Slides were then washed in 0.2x SSC for 10 sec and centrifuged dry at 500 rpm for 5 min. Measurement data and specifications: â?¢ Type of scanning hardware and software used: Microarrays were scanned using a GenePix 4000B scanner (Axon Instruments, Union City, CA). â?¢ Type of image analysis software used: Images were gridded and spots were quantified using GenePix Pro 4.0 software (Axon Instruments, Union City, CA). â?¢ A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis: The measurements made by GenePix Pro 4.0 are described in the sample files. For all hybridizations, the log2 normalized Median of Ratios (as described below) was used. â?¢ Data selection and transformation procedures: Arrays were auto-gridded by GenePix 4.0. Features flagged by GenePix as not found during grid
2010-06-05 | E-GEOD-6099 | biostudies-arrayexpress
Project description:The possibility of using quantum computers for electronic structure calculations has opened up a promising avenue for computational chemistry. Towards this direction, numerous algorithmic advances have been made in the last five years. The potential of quantum annealers, which are the prototypes of adiabatic quantum computers, is yet to be fully explored. In this work, we demonstrate the use of a D-Wave quantum annealer for the calculation of excited electronic states of molecular systems. These simulations play an important role in a number of areas, such as photovoltaics, semiconductor technology and nanoscience. The excited states are treated using two methods, time-dependent Hartree-Fock (TDHF) and time-dependent density-functional theory (TDDFT), both within a commonly used Tamm-Dancoff approximation (TDA). The resulting TDA eigenvalue equations are solved on a D-Wave quantum annealer using the Quantum Annealer Eigensolver (QAE), developed previously. The method is shown to reproduce a typical basis set convergence on the example [Formula: see text] molecule and is also applied to several other molecular species. Characteristic properties such as transition dipole moments and oscillator strengths are computed as well. Three potential energy profiles for excited states are computed for [Formula: see text] as a function of the molecular geometry. Similar to previous studies, the accuracy of the method is dependent on the accuracy of the intermediate meta-heuristic software called qbsolv.
| S-EPMC8458378 | biostudies-literature
Project description:The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.
| S-EPMC4490557 | biostudies-literature
Project description:Strong coupling between light and matter leads to the spontaneous formation of hybrid light-matter states, having different energies than the uncoupled states. This opens up for new ways of modifying the energy landscape of molecules without changing their atoms or structure. Heavy metal-free organic light emitting diodes (OLED) use reversed intersystem crossing (RISC) to harvest light from excited triplet states. This is a slow process, thus increasing the rate of RISC could potentially enhance OLED performance. Here we demonstrate selective coupling of the excited singlet state of Erythrosine B without perturbing the energy level of a nearby triplet state. The coupling reduces the triplet-singlet energy gap, leading to a four-time enhancement of the triplet decay rate, most likely due to an enhanced rate of RISC. Furthermore, we anticipate that strong coupling can be used to create energy-inverted molecular systems having a singlet ground and lowest excited state.
| S-EPMC5995866 | biostudies-literature