Conformational dynamics of the anticodon loop in yeast tRNAPhe as sensed by the fluorescence of wybutine

Conformational and dynamic properties of the anticodon loop of yeast tRNA^Phe were investigated by analyzing the time resolved fluorescence of wybutine serving as a local structural probe adjacent to the anticodon GmAA on its 3 side. The influence of Mg²⁺, important for stabilizing the tertiary structure of tRNA, and of the complementary anticodon s²UUC of E. coli tRNA ₂ ^Glu were investigated.Fluorescence lifetimes and anisotropies were measured with ps time resolution using time correlated single photon counting and a mode locked synchronously pumped and frequency doubled dye laser as excitation source. From the analysis of lifetimes () and rotational relaxation times ( _R ) we conclude that wybutine occurs in various structural states: (i) one stacked conformation where the base has no free mobility and the only rotational motion reflects the mobility of the whole tRNA molecule (=6 ns, _R =19 ns), (ii) an unstacked conformation where the base can freely rotate (=100 ps, _R = 370 ps) and (iii) an intermediary state (=2 ns, _R = 1.6 ns).Under biological conditions, i. e. in the presence of Mg²⁺ and neutral salts, wybutine is found in a stacked and immobile state which is consistent with the crystallographic picture. In the presence of the complementary codon however, as exemplified by the E. coli-tRNA ₂ ^Glu anticodon, our analysis indicates that the codon-anticodon complex exists in an equilibrium of structural states with different rotational mobility of wybutine. The conformation with wybutine freely mobile is the predominant one and suggests that this conformation of the codon-anticodon structure differs from the canonical 3–5 stack.     

Conformational Change of Chymotryptic Myosin Rod

Myosin rod was prepared from hen myosin by chymotryptic digestion. The indigested myosin was successfully removed by ultracentrifugation following alcohol treatment. No significant difference in UV absorption and CD spectra was observed between pH 7.0 and pH 10.5 for both myosin rod and myosin. When pH was raised to 11.7, the phenolic groups of the tyrosyl residues were ionized, and the helical configuration of the myosin rod and myosin could not withstand the electrostatic repulsion. When pH was further raised to 13.6, “abnormal” tyrosyl residues were ionized, resulting in decreased helix content. However, the myosin rod was stabler and less flexible against pH change than myosin, because of the lower content of tyrosyl residues in myosin rod.     

Transfer RNA Identity Change in Anticodon Variants of E. coli tRNAPhe in Vivo

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA^Phe. Constructing different anticodon mutants of E. coli tRNA^Phe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA^Phe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA^Phe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA^Phe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.     

Conformational Change of a Natto Mucin in Solution

The mucin obtained from a natto sample was found to be composed of 58 % of γ-polyglutamic acid and 40% of polysaccharide. The ratio of l- and d-glutamic acid was determined to be 58:42 using l-glutamic acid decarboxylase. The weight- and z-average molecular weight were 2.08 × 10⁵ and 2.22 × 10⁵, respectively. The distribution curve of the sedimentation coefficient showed a small heterogeneity. The mucin molecule was considered to be randomly coiled at pH 5.0 ~ 8.8 and to be a rod-like molecule in the lower pH region.     

Dissection of the ATP-Dependent Conformational Change Cycle of a Group II Chaperonin

Group II chaperonin captures an unfolded protein while in its open conformation and then mediates the folding of the protein during ATP-driven conformational change cycle. In this study, we performed kinetic analyses of the group II chaperonin from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TKS1-Cpn), by stopped-flow fluorometry and stopped-flow small-angle X-ray scattering to reveal the reaction cycle. Two TKS1-Cpn variants containing a Trp residue at position 265 or position 56 exhibit nearly the same fluorescence kinetics induced by rapid mixing with ATP. Fluorescence started to increase immediately after the start of mixing and reached a maximum at 1–2 s after mixing. Only in the presence of K⁺ that a gradual decrease in fluorescence was observed after the initial peak. Similar results were obtained by stopped-flow small-angle X-ray scattering. A rapid fluorescence increase, which reflects nucleotide binding, was observed for the mutant containing a Trp residue near the ATP binding site (K485W), irrespective of the presence or absence of K⁺. Without K⁺, a small, rapid fluorescence decrease followed the initial increase, and then a gradual decrease was observed. In contrast, with K⁺, a large, rapid fluorescence decrease occurred just after the initial increase, and then the fluorescence gradually increased. Finally, we observed ATP binding signal and also subtle conformational change in an ATPase-deficient mutant with K485W mutation. Based on these results, we propose a reaction cycle model for group II chaperonins.     

Measuring Integrin Conformational Change on the Cell Surface with Super-Resolution Microscopy

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Oligomeric-State-Dependent Conformational Change of the BLUF Protein TePixD (Tll0078)

The photochemical reaction dynamics of a BLUF (sensors of blue light using FAD) protein, PixD, from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD, Tll0078) were studied by pulsed laser-induced transient grating method. After the formation of an intermediate species with a red-shifted absorption spectrum, two new reaction phases reflecting protein conformational changes were discovered; one reaction phase manifested itself as expansion of partial molar volume with a time constant of 40 μs, whereas the other reaction phase represented a change in the diffusion coefficient D [i.e., the diffusion-sensitive conformational change (DSCC)]. D decreased from 4.9 × 10^− 11 to 4.4 × 10^− 11 m² s^− 1 upon the formation of the first intermediate, and subsequently showed a more pronounced decrease to 3.2 × 10^− 11 m² s^− 1 upon formation of the second intermediate. From a global analysis of signals at various grating wavenumbers, the time constant of D-change was determined to be 4 ms. Although the magnitude and rate constant of the faster volume change were independent of protein concentration, the amplitude of the signal that reflects the later DSCC significantly decreased as the protein concentration decreased. This concentration dependence suggests that two species exist in solution: a reactive species exhibiting the DSCC, and a second species that is nonreactive. The fraction of these species was found to be dependent on the concentration. The difference in reactivity was attributed to the different oligomeric states of TePixD (i.e., pentamer and decamer). The equilibrium of these states in the dark was confirmed by size-exclusion chromatography at various concentrations. These results demonstrated that only the decamer state is responsible for the conformational change. The results may suggest that the oligomeric state is functionally important in the signal transduction of this photosensory protein.     

Homology-Modelling Protein-Ligand Interactions: Allowing for Ligand-Induced Conformational Change

Current homology-modelling methods do not consider small molecules in their automated processes. Therefore, the development of a reliable tool for protein-ligand homology modelling is an important next step in generating plausible models for molecular interactions. Two automated protein-ligand homology-modelling strategies, requiring no expert knowledge from the user, are investigated here. Both employ the “induced fit” concept with flexibility in side chains and ligand. The most successful strategy superimposes the new ligand over the original ligand before homology modelling, allowing the new ligand to be taken into consideration during protein modelling (rather than after), facilitating conformational change in the local backbone if necessary. We show that this approach results in successful modelling of the ligand and key binding-site residues of angiotensin-converting enzyme 2 (ACE2) from its homologue ACE, which is not possible via conventional homology modelling or by homology modelling followed by docking. Several other difficult target complexes are also successfully modelled, reproducing native protein-ligand contacts with significantly different biological substrates and different binding-site conformations. These include the modelling of Cdk5 (cyclin-dependent kinase 5) from Cdk2, thymidine phosphorylase from a bacterial homologue, and dihydrofolate reductase from a recombinant variant with a markedly different inhibitor. In terms of average modelling quality across 82 targets, the ligand RMSD with respect to the experimental structure is 1.4 Å (and 2.0 Å for the protein binding site) for “easy” cases and 2.9 Å for the ligand (and 2.7 Å for the protein binding site) in “hard” cases. This demonstrates the importance of selecting an optimal template. Ligand-modelling accuracy is strongly dependent on target-template ligand structural similarity, rather than target-template sequence identity. However, protein-modelling accuracy is dependent on both. Our automated protein-ligand homology-modelling strategy generates a higher degree of accuracy than homology modelling followed by docking, generating an average ligand RMSD that is 1-2 Å better than docking with homology models.     

Statistical Assessment of Change Point Detectors for Single Molecule Kinetic Analysis

Change point detectors (CPDs) are used to segment recordings of single molecules for the purpose of kinetic analysis. The assessment of the accuracy of CPD algorithms has usually been based on testing them with simulated data. However, there have not been methods to assess the output of CPDs from real data independent of simulation. Here we present one method to do this based on the assumption that the elementary kinetic unit is a stationary period (SP) with a normal distribution of samples, separated from other SPs by change points (CPs). Statistical metrics of normality can then be used to assess SPs detected by a CPD algorithm (detected SPs, DSPs). Two statistics in particular were found to be useful, the z-transformed skew (S _Z) and z-transformed kurtosis (K _Z). K _Z(S _Z) plots of DSP from noise, simulated data and single ion channel recordings showed that DSPs with false negative CP could be distinguished. Also they showed that filtering had a significant effect on the normality of data and so filtering should be taken into account when calculating statistics. This method should be useful for analyzing single molecule recordings where there is no simple model for the data.     

Dependence of B-A Conformational Change in DNA on Base Composition

IT is generally accepted that all native DNAs, irrespective of their origin, give essentially identical X-ray diffraction patterns characteristic of the A form at high humidities and of the A form at lower humidities^{1, 2}. The purpose of the work described here was an accurate characterization of B-A conformational changes in a range of DNA by infrared dichroism, which seems to be particularly appropriate for the structural characterization of nucleic acids^3–6 as a complement of X-ray diffraction. In previous infrared studies the A form and B-A conformational changes were not observed⁷.     

Subwavelength Quarter-Waveplate Composed of L-Shaped Metal Nanoparticles

We propose a subwavelength quarter-waveplate composed of four L-shaped nanoparticles that act as optical nanoantennas and investigate its optical properties using the finite-difference time-domain method. When polarization of the incident beam is parallel to one arm of the antenna, polarization of the scattering light rotates 45° with respect to that of the incident beam due to the symmetry of the L-shaped nanoantennas. Phase retardation is generated by changing the arm length of the antennas. The influence of the distance between the L-shaped nanoantennas on the far-field radiation is also discussed.     

Low pH-Induced Conformational Change in Herpes Simplex Virus Glycoprotein B

Herpesviruses can enter host cells using pH-dependent endocytosis pathways in a cell-specific manner. Envelope glycoprotein B (gB) is conserved among all herpesviruses and is a critical component of the complex that mediates membrane fusion and entry. Here we demonstrate that mildly acidic pH triggers specific conformational changes in herpes simplex virus (HSV) gB. The antigenic structure of gB was specifically altered by exposure to low pH both in vitro and during entry into host cells. The oligomeric conformation of gB was altered at a similar pH range. Exposure to acid pH appeared to convert virion gB into a lower-order oligomer. The detected conformational changes were reversible, similar to those in other class III fusion proteins. Exposure of purified, recombinant gB to mildly acidic pH resulted in similar changes in conformation and caused gB to become more hydrophobic, suggesting that low pH directly affects gB. We propose that intracellular low pH induces alterations in gB conformation that, together with additional triggers such as receptor binding, are essential for virion-cell fusion during herpesviral entry by endocytosis.Herpes simplex virus (HSV) is an important human pathogen, causing significant morbidity and mortality worldwide. HSV enters host cells by fusion of the viral envelope with either an endosomal membrane (38) or the plasma membrane (63). The entry pathway taken is thought to be determined by both virus (17, 45) and host cell (4, 17, 35, 39, 45) factors. Based on experiments with lysosomotropic agents, which elevate the normally low pH of endosomes, acidic pH has been implicated in the endocytic entry of HSV into several cell types, including human epithelial cells (37). Low pH has also recently been implicated in cell infection by several other human and veterinary herpesviruses (1, 21, 26, 47). The mechanistic role of endosomal pH in herpesvirus entry into cells is not known.Herpesviruses are a paradigm for membrane fusion mediated by a complex of several glycoproteins. We have proposed that HSV likely encodes machinery to mediate both pH-dependent and pH-independent membrane fusion reactions. Envelope glycoproteins glycoprotein B (gB) and gD and the heterodimer gH-gL are required for both pH-independent and pH-dependent entry pathways (11, 22, 30, 39, 46). Interaction of gD with one of its cognate receptors is an essential trigger for membrane fusion and entry (13, 52), regardless of the cellular pathway. However, engagement of a gD receptor is not sufficient for fusion, and at least one additional unknown trigger involving gB or gH-gL is likely necessary. gB is conserved among all herpesviruses, and in all cases studied to date, it plays roles in viral entry, including receptor binding and membrane fusion. The crystal structure of an ectodomain fragment of HSV type 1 (HSV-1) gB is an elongated, rod-like structure containing hydrophobic internal fusion loops (28). This structure bears striking architectural homology to the low pH, postfusion form of G glycoprotein from vesicular stomatitis virus (VSV-G) (43). Both the gB and G structures have features of class I and class II fusion proteins and are thus designated class III proteins (57).During entry of the majority of virus families, low pH acts directly on glycoproteins to induce membrane fusion (60). In some cases, the low pH trigger is not sufficient, or it plays an indirect role. For example, host cell proteases, such as cathepsins D and L, require intravesicular low pH to cleave Ebola virus and severe acute respiratory syndrome (SARS) glycoproteins to trigger fusion (14, 51).We investigated the role of low pH in the molecular mechanism of herpesviral entry. The results suggest that mildly acidic pH, similar to that found within endosomes, triggers a conformational change in gB. We propose that, together with other cellular cues such as receptor interaction, intracellular low pH can play a direct activating role in HSV membrane fusion and entry.     

Codon-induced association of the isolated anticodon loop of tRNAPhe

The binding of the codon UUC to the isolated anticodon loop of tRNAPhe (yeast) has been studied as a model of codon recognition by a simple adaptor. Fluorescence titrations demonstrate that UUC binds to the isolated anticodon loop with an equilibrium constant of 1.4 X 10(3) M-1 (at 7.2 degrees C). Equilibrium sedimentation curves reveal that UUC binding induces association of anticodon loops beyond the dimer stage. A set of complete sedimentation curves obtained for various reactant concentrations was analyzed according to a model with an infinite number of subsequent association steps for UUC-anticodon loop complexes and with equal affinity for each step. The coupling of association and sedimentation was considered quantitatively, and the information resulting from conservation of mass was used by integration. According to this procedure, the experimental data can be described by an isodesmic association constant of 8 X 10(3) M-1 with satisfactory accuracy. Temperature-jump relaxation detected by fluorescence measurements provides independent evidence for codon-induced association of the anticodon loop. The data are consistent with the following mechanism: UUC preferentially binds to one of two loop conformations with a rate constant of 4.5 X 10(6) M-1 s-1; the UUC-anticodon loop complex undergoes association with a rate constant of 6.5 X 10(6) M-1 s-1. The reactions observed for the isolated anticodon loop are surprisingly similar to those observed previously for the complete tRNA, suggesting that simple hairpin loops are appropriate adaptors for a translation process at an early stage of evolution; the codon-induced association of the hairpin loop should be very useful to facilitate the transfer of cognate amino acids during translation.     

Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding

The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.