Cooperative Conformational Changes Research Articles

Cooperative Conformational Transitions Underpin the Activation Heat Capacity in the Temperature Dependence of Enzyme Catalysis.

Many enzymes display non-Arrhenius behavior with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behavior and recently the role of the activation heat capacity (ΔCP⧧) has been widely discussed. If enzyme-catalyzed reactions occur with appreciable negative values of ΔCP⧧ (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalyzed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterize the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallized the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 Å). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalizes disparate behavior seen for MalL and previous results for a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterize the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilization that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity and other aspects of enzyme catalysis.

The structure of NAD+ consuming protein Acinetobacter baumannii TIR domain shows unique kinetics and conformations

Toll-like and interleukin-1/18 receptor/resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD+ hydrolysis and map the conformational effects of its interaction with NAD+ using hydrogen-deuterium exchange-mass spectrometry. NAD+ results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes, which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using the minimally invasive spectroscopic method 2-photon excitation with fluorescence lifetime imaging, which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIRE208A protein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.

Sampling of Protein Conformational Space Using Hybrid Simulations: A Critical Assessment of Recent Methods.

Recent years have seen several hybrid simulation methods for exploring the conformational space of proteins and their complexes or assemblies. These methods often combine fast analytical approaches with computationally expensive full atomic molecular dynamics (MD) simulations with the goal of rapidly sampling large and cooperative conformational changes at full atomic resolution. We present here a systematic comparison of the utility and limits of four such hybrid methods that have been introduced in recent years: MD with excited normal modes (MDeNM), collective modes-driven MD (CoMD), and elastic network model (ENM)-based generation, clustering, and relaxation of conformations (ClustENM) as well as its updated version integrated with MD simulations (ClustENMD). We analyzed the predicted conformational spaces using each of these four hybrid methods, applied to four well-studied proteins, triosephosphate isomerase (TIM), 3-phosphoglycerate kinase (PGK), HIV-1 protease (PR) and HIV-1 reverse transcriptase (RT), which provide extensive ensembles of experimental structures for benchmarking and comparing the methods. We show that a rigorous multi-faceted comparison and multiple metrics are necessary to properly assess the differences between conformational ensembles and provide an optimal protocol for achieving good agreement with experimental data. While all four hybrid methods perform well in general, being especially useful as computationally efficient methods that retain atomic resolution, the systematic analysis of the same systems by these four hybrid methods highlights the strengths and limitations of the methods and provides guidance for parameters and protocols to be adopted in future studies.

Cooperative Conformational Change of Bio-Inspired Organic Molecule for Ultrafast Lithium-Ion Batteries

Biological energy transduction occurs through stepwise redox reactions of various organic molecules, such as adenosine triphosphate, quinone derivatives, nicotinamide and flavin cofactors.1,2 The redox reactions in the biological system have inspired the design of biomimetic materials for renewable energy and environmental science.3-5 Recently, the exploitation of biological redox reactions has provided a new approach to the design of organic electrode materials for rechargeable batteries.6,7 Redox-active bioorganic molecules led to the development of high-performance battery systems owing to their reversible redox activities.8 For examples, quinone and flavin derivatives, in which stable redox states exist during photosynthesis and cellular respiration, showed reversible battery operation at ~2.5 V vs. Li+/Li.9,10 Electron-withdrawing groups (e.g. N and O) in the vicinity of the redox active centers (e.g. C=O and C=N) in quinone and flavin derivatives further elevated the redox potential by attracting the electrons from the electron cloud around the redox active centers. Unfortunately, battery performance of organic-based electrodes is still lacking of energy and power density to be implemented into the practical energy storage system.11 To tackle this issue, the organic molecules with more redox moieties and higher conductivity have been explored.12,13 In addition, to improve the stability of organic molecules as well as inter-charge transfer, nano-dimensional networking between carbon conductor and organic molecules is of great interest so far.14,15 However, the organic-battery performance can be influenced by molecular geometry and molecular orbital structure according to the literature.16,17 Therefore, we envisage the cooperative conformational change of organic molecules during battery operation.Herein, as depicted in Figure 1, we unveiled that the proton-coupled redox reaction of single organic molecule in an aqueous solution can be translated to the lithium-coupled redox reaction of single organic molecule in a lithium-based organic electrolyte by using phenoxazin-3-one (i.e., resorufin) as a new bio-inspired redox-active molecule. In this report, we elaborate the cooperative conformational change of the single molecule during the redox reaction, which ultimately lead to the outstanding battery performance for the first time. Phenoxazin-3-one is selected as the model single organic molecule. Our analyses using operando Raman spectroscopy and DFT calculations confirmed the conformational flexibility in the molecular shape, enabling formation of strong p-p interactions between redox-active organic molecule and carbon. The strong p-p interaction of the single organic molecule with carbon resulted in the excellent battery performance (high discharge capacity, 298 mAhg-1, and outstanding rate capability, 65% retention at 10 C). Our work provides in-depth understanding about cooperative conformational change in the single molecule and its effect on the battery performance. Furthermore, we suggest a phenoxazin-3-one as a new redox-active molecules derived from biochemical redox reaction for energy storage materials in Li-ion rechargeable batteries.(1) B. B. Lowell, B. M. Spiegelman, Nature 2000, 404, 652.(2) H. Wang, Y. Yang, L. Guo, Adv. Energy Mater. 2017, 7, 1700663.(3) L. Que Jr, W. B. Tolman, Nature 2008, 455, 333.(4) U. G. K. Wegst, H. Bai, E. Saiz, A. P. Tomsia, R. O. Nat. Mater. 2014, 14, 23.(5) Y. J. Lee, H. Yi, W.-J. Kim, K. Kang, D. S. Yun, M. S. Strano, G. Ceder, A. M. Belcher, Science 2009, 324, 1051.(6) Y. Ding, Y. Li, G. Yu, Chem 2016, 1, 790.(7) B. Lee, Y. Ko, G. Kwon, S. Lee, K. Ku, J. Kim, K. Kang, Joule 2018, 2, 61.(8) J. Hong, M. Lee, B. Lee, D.-H. Seo, C. B. Park, K. Kang, Nat. Commun. 2014, 5, 5335.(9) M. Lee, J. Hong, D.-H. Seo, D. H. Nam, K. T. Nam, K. Kang, C. B. Park, Angew. Chem. Int. Ed. 2013, 52, 8322.(10) Z. Song, Y. Qian, X. Liu, T. Zhang, Y. Zhu, H. Yu, M. Otani, H. Zhou, Energy Environ. Sci. 2014, 7, 4077.(11) S. Lee, G. Kwon, K. Ku, K. Yoon, S.-K. Jung, H.-D. Lim, K. Kang, Adv. Mater. 2018, 30, 1704682.(12) B. Häupler, A. Wild, U. S. Schubert, Adv. Energy Mater. 2015, 5, 1402034.(13) Q. Zhao, Y. Lu, J. Chen, Adv. Energy Mater. 2017, 7, 1601792.(14) M. Lee, J. Hong, H. Kim, H.-D. Lim, S. B. Cho, K. Kang, C. B. Park, Adv. Mater. 2014, 26, 2558.(15) C. Luo, R. Huang, R. Kevorkyants, M. Pavanello, H. He, C. Wang, Nano Lett. 2014, 14, 1596(16) Y. Morita, S. Nishida, T. Murata, M. Moriguchi, A. Ueda, M. Satoh, K. Arifuku, K. Sato, T. Takui, Nat. Mater. 2011, 10, 947.(17) V. A. Mihali, S. Renault, L. Nyholm, D. Brandell, RSC Adv. 2014, 4, 38004. Figure 1

Neutralization of the anthrax toxin by antibody-mediated stapling of its membrane-penetrating loop.

Anthrax infection is associated with severe illness and high mortality. Protective antigen (PA) is the central component of the anthrax toxin, which is one of two major virulence factors of Bacillus anthracis, the causative agent of anthrax disease. Upon endocytosis, PA opens a pore in the membranes of endosomes, through which the cytotoxic enzymes of the toxin are extruded. The PA pore is formed by a cooperative conformational change in which the membrane-penetrating loops of PA associate, forming a hydrophobic rim that pierces the membrane. Due to its crucial role in anthrax progression, PA is an important target for monoclonal antibody-based therapy. cAb29 is a highly effective neutralizing antibody against PA. Here, the cryo-EM structure of PA in complex with the Fab portion of cAb29 was determined. It was found that cAb29 neutralizes the toxin by clamping the membrane-penetrating loop of PA to the static surface-exposed loop of the D3 domain of the same subunit, thereby preventing pore formation. These results provide the structural basis for the antibody-based neutralization of PA and bring into focus the membrane-penetrating loop of PA as a target for the development of better anti-anthrax vaccines.

Cooperative Conformational Change of a Single Organic Molecule for Ultrafast Rechargeable Batteries

We unveil that the conformational change of a single organic molecule during the redox reaction leads to impressive battery performance for the first time. We propose the model material, a phenoxaz...

Long-Range and Directional Allostery of Actin Filaments Plays Important Roles in Various Cellular Activities.

A wide variety of uniquely localized actin-binding proteins (ABPs) are involved in various cellular activities, such as cytokinesis, migration, adhesion, morphogenesis, and intracellular transport. In a micrometer-scale space such as the inside of cells, protein molecules diffuse throughout the cell interior within seconds. In this condition, how can ABPs selectively bind to particular actin filaments when there is an abundance of actin filaments in the cytoplasm? In recent years, several ABPs have been reported to induce cooperative conformational changes to actin filaments allowing structural changes to propagate along the filament cables uni- or bidirectionally, thereby regulating the subsequent binding of ABPs. Such propagation of ABP-induced cooperative conformational changes in actin filaments may be advantageous for the elaborate regulation of cellular activities driven by actin-based machineries in the intracellular space, which is dominated by diffusion. In this review, we focus on long-range allosteric regulation driven by cooperative conformational changes of actin filaments that are evoked by binding of ABPs, and discuss roles of allostery of actin filaments in narrow intracellular spaces.

Solvent flows, conformation changes and lattice reordering in a cold protein crystal.

When protein crystals are abruptly cooled, the unit-cell, protein and solvent-cavity volumes all contract, but the volume of bulk-like internal solvent may expand. Outflow of this solvent from the unit cell and its accumulation in defective interior crystal regions has been suggested as one cause of the large increase in crystal mosaicity on cooling. It is shown that when apoferritin crystals are abruptly cooled to temperatures between 220 and 260 K, the unit cell contracts, solvent is pushed out and the mosaicity grows. On temperature-dependent timescales of 10 to 200 s, the unit-cell and solvent-cavity volume then expand, solvent flows back in, and the mosaicity and B factor both drop. Expansion and reordering at fixed low temperature are associated with small-amplitude but large-scale changes in the conformation and packing of apoferritin. These results demonstrate that increases in mosaicity on cooling arise due to solvent flows out of or into the unit cell and to incomplete, arrested relaxation of protein conformation. They indicate a critical role for time in variable-temperature crystallographic studies, and the feasibility of probing interactions and cooperative conformational changes that underlie cold denaturation in the presence of liquid solvent at temperatures down to ∼200 K.

Probing the Behaviour of Cas1-Cas2 upon Protospacer Binding in CRISPR-Cas Systems using Molecular Dynamics Simulations

Adaptation in CRISPR-Cas systems enables the generation of an immunological memory to defend against invading viruses. This process is driven by foreign DNA spacer (termed protospacer) selection and integration mediated by Cas1-Cas2 protein. Recently, different states of Cas1-Cas2, in its free form and in complex with protospacer DNAs, were solved by X-ray crystallography. In this paper, molecular dynamics (MD) simulations are employed to study crystal structures of one free and two protospacer-bound Cas1-Cas2 complexes. The simulated results indicate that the protospacer binding markedly increases the system stability, in particular when the protospacer containing the PAM-complementary sequence. The hydrogen bond and binding free energy calculations explain that PAM recognition introduces more specific interactions to increase the cleavage activity of Cas1. By using principal component analysis (PCA) and intramolecular angle calculation, this study observes two dominant slow motions associated with the binding of Ca1-Cas2 to the protospacer and potential target DNAs respectively. The comparison of DNA structural deformation further implies a cooperative conformational change of Cas1-Cas2 and protospacer for the target DNA capture. We propose that this cooperativity is the intrinsic requirement of the CRISPR integration complex formation. This study provides some new insights into the understanding of CRISPR-Cas adaptation.

Environmental adjustments of the cooperativity in M13 phage thermal denaturation

The filamentous bacteriophage M13 has been widely used in phage display and as a scaffold for nanostructures. Although M13 is loosely described as a thermo-resistant virus, no thorough studies of its thermostability have been reported so far. By using differential scanning calorimetry (DSC), we characterized the thermal stability of M13 in aqueous buffer and in the presence of ethanol. M13 was found to be very thermostable indeed, being resistant to the combined effects of temperature and high ethanol content. Upon thermal denaturation, several subunits of the major coat protein apparently undergo a cooperative conformational change, leading to the formation of residual β-strand conformation. Furthermore, the cooperativity of the denaturation process changes drastically with pH and ethanol concentration, indicating that P8 moieties are able to adjust its intermolecular packing in response to the environment. Deconvolution of DSC scans allowed the estimation of the number of P8 molecules comprising one cooperative unit.

The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments

Cofilin/ADF proteins are actin-remodeling proteins, essential for actin disassembly in various cellular processes, including cell division, intracellular transport, and motility. Cofilins bind actin filaments cooperatively and sever them preferentially at boundaries between bare and cofilin-decorated (cofilactin) segments. The cooperative binding to actin has been proposed to originate from conformational changes that propagate allosterically from clusters of bound cofilin to bare actin segments. Estimates of the lengths over which these cooperative conformational changes propagate vary dramatically, ranging from 2 to >100 subunits. Here, we present a general, structure-based method for detecting from cryo-EM micrographs small variations in filament geometry (i.e. twist) with single-subunit precision. How these variations correlate with regulatory protein occupancy reveals how far allosteric, conformational changes propagate along filaments. We used this method to determine the effects of cofilin on the actin filament twist. Our results indicate that cofilin-induced changes in filament twist propagate only 1-2 subunits from the boundary into the bare actin segment, independently of the boundary polarity (i.e. irrespective of whether or not the bare actin segment flanks the pointed or barbed-end side of the boundary) and the pyrene fluorophore labeling of actin. These observations indicate that the filament twist changes abruptly at boundaries between bare and cofilin-decorated segments, thereby constraining mechanistic models of cooperative actin filament interactions and severing by cofilin. The methods presented here extend the capability of cryo-EM to analyze biologically relevant deviations from helical symmetry in actin as well as other classes of linear polymers.

Unidirectional growth of heavy meromyosin clusters along actin filaments revealed by real-time fluorescence microscopy.

Heavy meromyosin (HMM) forms clusters along actin filaments under low ATP concentrations. Here, we observed the growth of HMM clusters under low concentrations of ATP in real time using fluorescence microscopy. When actin filaments were loosely immobilized on positively charged lipid bilayers, clusters of HMM-GFP were readily formed. Time-lapse observation revealed that the clusters grew unidirectionally. When we used a mixture of actin filaments and copolymers of actin and acto-S1dC, a chimeric protein of actin and the myosin motor domain, HMM-GFP preferentially formed clusters along the copolymers. We thus suggest that binding of myosin motors carrying ADP and Pi induces unidirectional conformational changes in actin filaments and allosterically recruits more myosin binding. In contrast, when actin filaments and copolymers were anchored to glass substrate via stable biotin-avidin linkage, higher concentrations of HMM-GFP were required to form clusters than on the lipid bilayer. Moreover, actin filaments and copolymers were not discriminated regarding preferential cluster formation. This is presumably because the myosin-induced cooperative conformational changes in actin filaments involve changes in the helical twist. Consistent with this, cofilin clusters, which supertwist the helix, were readily formed along loosely immobilized actin filaments, but not along those anchored via biotin-avidin linkage.

Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

Secondary Structural Change Can Occur Diffusely and Not Modularly during Protein Folding and Unfolding Reactions.

A major goal of protein folding studies is to understand the structural basis of the coupling between stabilizing interactions, which leads to cooperative conformational change. The goal is challenging because of the difficulty in simultaneously measuring global cooperativity by determining population distributions of the conformations present, and the structures of these conformations. Here, hydrogen exchange (HX) into the small protein monellin was carried out under conditions where structure-opening is rate limiting for most backbone amide sites. Detection by mass spectrometry allowed characterization of not only segment-specific structure-opening rates but also the cooperativity of unfolding of the different secondary structural segments of the protein. The segment-specific pattern of HX reveals that the backbone hydrogen-bonding network disassembles in a structurally diffuse, asynchronous manner. A comparison of the site-specific transient opening rates of secondary and tertiary structure in the protein provides a structural rationale for the observation that unfolding is hierarchical and describable by exponential kinetics, despite being diffuse. Since unfolding was studied in native conditions, the sequence of events during folding in the same conditions will be the reverse of the sequence of events observed during unfolding. Hence, the formation of secondary structural units during folding would also occur in a non-cooperative, diffuse, and asynchronous manner.

Cofilin-induced cooperative conformational changes of actin subunits revealed using cofilin-actin fusion protein.

To investigate cooperative conformational changes of actin filaments induced by cofilin binding, we engineered a fusion protein made of Dictyostelium cofilin and actin. The filaments of the fusion protein were functionally similar to actin filaments bound with cofilin in that they did not bind rhodamine-phalloidin, had quenched fluorescence of pyrene attached to Cys374 and showed enhanced susceptibility of the DNase loop to cleavage by subtilisin. Quantitative analyses of copolymers made of different ratios of the fusion protein and control actin further demonstrated that the fusion protein affects the structure of multiple neighboring actin subunits in copolymers. Based on these and other recent related studies, we propose a mechanism by which conformational changes induced by cofilin binding is propagated unidirectionally to the pointed ends of the filaments, and cofilin clusters grow unidirectionally to the pointed ends following this path. Interestingly, the fusion protein was unable to copolymerize with control actin at pH 6.5 and low ionic strength, suggesting that the structural difference between the actin moiety in the fusion protein and control actin is pH-sensitive.

Energetics of LCST transition of poly(ethylene oxide) in aqueous solutions

Poly(ethylene oxide), PEO, is known to undergo a phase separation transition in aqueous solutions upon heating. This LCST transition is first investigated by high-sensitivity differential scanning calorimetry. Dependences of the transition temperature (Tt), enthalpy (Δth) and heat capacity increment (Δtcp) on the polymer concentration are determined. The transition temperature and enthalpy of PEO decreased with increasing the polymer concentration and followed the Kirchhoff law. A substantial positive value of the transition heat capacity increment of PEO is detected suggesting that the transition is accompanied by an exposure of the polymer hydrophobic groups to water. Comparison of Δtcp with the corresponding change in the accessible surface area of PEO reveals that the LCST transition of PEO may involve cooperative conformational changes of the type of helix–coil transition in PEO macromolecules.

Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy.

High-speed atomic force microscopy was employed to observe structural changes in actin filaments induced by cofilin binding. Consistent with previous electron and fluorescence microscopic studies, cofilin formed clusters along actin filaments, where the filaments were 2-nm thicker and the helical pitch was ~25% shorter, compared to control filaments. Interestingly, the shortened helical pitch was propagated to the neighboring bare zone on the pointed-end side of the cluster, while the pitch on the barbed-end side was similar to the control. Thus, cofilin clusters induce distinctively asymmetric conformational changes in filaments. Consistent with the idea that cofilin favors actin structures with a shorter helical pitch, cofilin clusters grew unidirectionally toward the pointed-end of the filament. Severing was often observed near the boundaries between bare zones and clusters, but not necessarily at the boundaries.

The ribosome uses cooperative conformational changes to maximize and regulate the efficiency of translation.

One of the most challenging unanswered questions regarding the structural biology of biomolecular machines such as the two-subunit ribosome is whether and how these machines coordinate seemingly independent and random conformational fluctuations to maximize and regulate their functional efficiencies. To address this question, we have used ribosome mutagenesis or a ribosome-targeting antibiotic to predictably perturb the dynamics of intersubunit rotation, a structural rearrangement of the ribosome that is essential for the translocation and ejection of ribosome-bound tRNAs during translation. Concomitantly, we have used single-molecule fluorescence resonance energy transfer (smFRET) to characterize the effects of these perturbations on the dynamics of ribosomal L1 stalk movements and ribosome-bound tRNA reconfigurations, conformational changes that are likewise essential for the translocation and ejection of tRNAs during translation. Together with the results of complementary biochemical studies, our smFRET studies demonstrate that the ribosome uses cooperative conformational changes to maximize and regulate the efficiency with which it translocates and ejects tRNAs during translation. We propose that the ribosome employs cooperative conformational changes to efficiently populate global conformational states that are productive for translation, that translation factors exploit this cooperativity as part of their mechanisms of action, and that antibiotics exploit it to maximize the potency with which they inhibit translation. It is likely that similar cooperative conformational changes underlie the function and regulation of other biomolecular machines.

Cooperative Conformational Change and Excitation Migration of Biphenyl-PMO Amorphous Film, As Revealed by Femtosecond Time-Resolved Spectroscopy

Excited state dynamics of biphenyl-bridged mesoporous organosilica (Bp-PMO) film was investigated by femtosecond transient absorption and dichroism measurements at various excitation intensities. Under the excitation condition with low intensity (ca. 0.1 μJ/pulse), the relaxation from the excited Franck–Condon state with skewed structure of the two phenyl rings to the preplanar state occurred with a time constant of 550 fs, followed by the excimer formation with two time constants of 9.0 and 140 ps. Under higher excitation condition with 1.0 μJ/pulse, very rapid excimer formation within 500 fs was observed. From the analysis of the transient absorption spectra, it was revealed that the cooperative geometrical relaxation from skewed to planar structures, in addition to the energy migration, led to the rapid excimer formation under the high excitation condition. By integrating these results with the fluorescence dynamics, the photoprimary processes in Bp-PMO, such as energy migration, annihilation, and excimer formation, were discussed.

The Ribosome Uses Cooperative Conformational Changes to Maximize the Efficiency of Protein Synthesis

Execution of individual steps in the multi-step functional cycles of large, multi-component molecular machines such as the ribosome almost universally requires remodeling of multiple, spatially distant structural components of the machine. In order to function efficiently, therefore, molecular machines likely must allosterically coordinate numerous, seemingly independent conformational rearrangements. Due to the significant technical challenges associated with characterizing their structural dynamics, however, the questions of whether and how large molecular machines coordinate such dynamics so as to maximize the efficiency with which they perform their biological functions remain exceptionally challenging to answer. Using a combination of structural and phylogenetic analyses, molecular genetics, single-molecule fluorescence resonance energy transfer, and in vitro biochemical assays, here we demonstrate that the ribosome uses cooperative conformational changes to maximize the efficiency with which it translocates and ejects its transfer RNA adaptors during protein synthesis. Interpretation of our data within the context provided by atomic-resolution ribosome structures and phylogenetic analyses of ribosomal RNA and ribosomal protein sequences leads us to propose a structure-based model for the observed cooperativity. Our results demonstrate that large, multi-component, molecular machines such as the ribosome can use networks of cooperative conformational changes to facilitate mechanical processes that would otherwise limit their catalytic rates.