Fast Dissociation Process Research Articles

Overlapping characteristics of weak interactions of two transcriptional regulators with WDR5

The WD40 repeat protein 5 (WDR5) is a nuclear hub that critically influences gene expression by interacting with transcriptional regulators. Utilizing the WDR5 binding motif (WBM) site, WDR5 interacts with the myelocytomatosis (MYC), an oncoprotein transcription factor, and the retinoblastoma-binding protein 5 (RbBP5), a scaffolding element of an epigenetic complex. Given the clinical significance of these protein-protein interactions (PPIs), there is a pressing necessity for a quantitative assessment of these processes. Here, we use biolayer interferometry (BLI) to examine interactions of WDR5 with consensus peptide ligands of MYC and RbBP5. We found that both interactions exhibit relatively weak affinities arising from a fast dissociation process. Remarkably, live-cell imaging identified distinctive WDR5 localizations in the absence and presence of full-length binding partners. Although WDR5 tends to accumulate within nucleoli, WBM-mediated interactions with MYC and RbBP5 require their localization outside nucleoli. We utilize fluorescence resonance energy transfer (FRET) microscopy to confirm these weak interactions through a low FRET efficiency of the MYC-WDR5 and RbBP5-WDR5 complexes in living cells. In addition, we evaluate the impact of peptide and small-molecule inhibitors on these interactions. These outcomes form a fundamental basis for further developments to clarify the multitasking role of the WBM binding site of WDR5.

Imaging the Photodissociation Dynamics and Fragment Alignment of CH2BrI at 193 nm.

The photodissociation dynamics and photofragment alignment of bromoiodomethane (CH2BrI) have been studied at 193 nm using a double experimental and theoretical approach. In addition, the ultraviolet (UV)-vacuum ultraviolet (VUV) absorption spectrum of gas phase CH2BrI has been measured in the photon energy range of 5-11 eV using the VUV Fourier transform spectrometer (FTS) at the VUV beamline DESIRS of the synchrotron SOLEIL facility. The slice imaging technique in combination with resonance enhanced multiphoton ionization (REMPI) detection of the Br(2PJ) and I(2PJ) (with J = 3/2 and 1/2 for Br/I and Br*/I*, respectively) atomic photofragments have been used to produce experimental translational energy and angular distributions, which were analyzed to deliver, on one hand, the partitioning of the available energy among the different degrees-of-freedom of the photofragments and, on the other, the photofragment polarization in terms of aqk(p) alignment parameters. The experimental measurements were rationalized in terms of high-level ab initio calculations of vertical excitation energies, transition dipole moments and potential energy curves (PECs) along different reaction coordinates to provide a complete picture of the photodissociation dynamics. The results indicate that for excitation at 193 nm, prompt C-X cleavage (with X being either halogen atom, Br or I) competes with fast internal conversion and consequent stochastic dissociation in lower electronic states. In the case of the CH2Br + I(2P3/2)/I*(2P1/2) channels, the dynamics are greatly biased toward the stochastic dissociation process due to both the particular PECs landscape and the unfavored excitation of the CH2BrI ensemble with respect to the C-I molecular axis at this excitation energy. The ab initio PECs provide a tentative path for the fast dissociation process in either case. For the C-Br bond breakage, excitation to the 13A' electronic state and predissociation through the 11A'/11A″ or 12A'/12A″ states, leading to direct dissociation through the 10A'/9A″ states, appear as the most consistent dynamics. For the C-I channel, predissociation does not become a reliable possibility and a fast internal conversion may precede dissociation through the repulsive 6A'/6A″ and 4A'/4A″ states. The large content of rotational and vibrational excitation of the polyatomic cofragments is justified through the soft impulsive model and the geometrical changes produced along the dissociation pathway. Strikingly, the aqk(p) alignment parameters obtained for the Br(2P3/2) and I(2P3/2) photoproducts indicate that the rotational angular momentum of the CH2X (X = I or Br) cofragment appears highly constrained along the recoil direction. Finally, this work presents a highly plausible explanation for the branching ratio of secondary dissociation processes in the photodynamics of CH2BrI at 193 nm.

Ion Imaging of MgI+ Photofragment in Ultraviolet Photodissociation of Mass-Selected Mg+ICH3 Complex.

We have observed images of MgI+ fragment ions produced in ultraviolet laser photodissociation of mass-selected Mg+ICH3 ions at 266 nm. Split distribution almost perpendicular to the polarization direction of the photolysis laser was observed in the photofragment image. Potential energy curves of Mg+ICH3 were obtained by theoretical calculations. Among these curves, the excited complex ion dissociated along almost repulsive potentials with several avoided crossings, which was connected to MgI+ + CH3. In the ground state of Mg+ICH3, the CH3I was bonded with Mg from the iodine side, and the Mg-I-C bond angle was calculated to be 101.1°. The theoretical results also indicated that the dissociation occurred after the 52A' ← 12A' photoexcitation, where the transition dipole moment was almost parallel to the Mg-I bond axis. The MgI+ and CH3 fragments dissociated each other parallel to the direction connecting those center-of-masses, which was 67° with respect to the transition dipole moment of 52A' ← 12A' photoexcitation. Therefore, the fragment recoil direction was assumed to approach perpendicular tendency against the polarization direction under the fast dissociation process. However, calculated potential energy curves showed a complicated reaction pathway for MgI+ production, including nonadiabatic processes, although the experimental results indicated the fast dissociation reaction.

Role of ultrafast dissociation in the fragmentation of chlorinated methanes.

Photon-induced fragmentation of a full set of chlorinated methanes (CH3Cl, CH2Cl2, CHCl3, CCl4) has been investigated both experimentally and computationally. Using synchrotron radiation and electron-ion coincidence measurements, the dissociation processes were studied after chlorine 2p electron excitation. Experimental evidence for CH3Cl and CH2Cl2 contains unique features suggesting that fast dissociation processes take place. By contrast, CHCl3 and CCl4 molecules do not contain the same features, hinting that they experience alternative mechanisms for dissociation and charge migration. Computational work indicates differing rates of charge movement after the core-excitation, which can be used to explain the differences observed experimentally.

Phase dependence of OD+, HOD+, and H3+ ions released from the deuterated dication of methanol under ω/2ω laser field irradiation

Multielectron dissociative ionization of gas phase deuterated methanol (CH3OD) molecules under the irradiation of a 30fs asymmetric two color (ω/2ω) laser field is studied by means of time-of-flight mass spectrometry. The dependence on phase φ between the two laser beams of OD+, HOD+, H3+ fragment ions released from the dissociation of the dication has been investigated. Based on the phase dependence and the recorded angular distributions, it is proposed that the OD+, HOD+ ions can be ejected from fast and slow dissociation processes. The fast dissociation process is attributed to fragmentation from singlet states. The possibility that the slow dissociation process involves a long-lived metastable state and/or dissociation from triplet states is also discussed. The dependence of the H3+ ion on phase φ reveals the existence of two different, as far as the time scale is concerned, stages in its production.

Determination of Peptide Topology through Time-Resolved Double-Resonance under Electron Capture Dissociation Conditions

Characterizing the conformation of biomolecules by mass spectrometry still represents a challenge. With their knotted structure involving a N-terminal macrolactam ring where the C-terminal tail of the peptide is threaded and sterically trapped, lasso peptides constitute an attractive model for developing methods for characterizing gas-phase conformation, through comparison with their unknotted topoisomers. Here, the kinetics of electron capture dissociation (ECD) of a lasso peptide, capistruin, was investigated by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry and compared to that of its branched-cyclic topoisomer, lactam-capistruin. Both peptides produced rather similar ECD spectra but showed different extent of H(•) transfer from c(i)' to z(j)(•) ions. Time-resolved double-resonance experiments under ECD conditions were performed to measure the formation rate constants of typical product ions. Such experiments showed that certain product ions, in particular those related to H(•) transfer, proceeded through long-lived complexes for capistruin, while fast dissociation processes predominated for lactam-capistruin. The formation rate constants of specific ECD product ions enabled a clear differentiation of the lasso and branched-cyclic topoisomers. These results indicate that the formation kinetics of ECD product ions constitute a new way to explore the conformation of biomolecules and distinguish between topoisomers and, more generally, conformers.

Imaging CH3SH photodissociation at 204 nm: the SH + CH3 channel

The SH + CH(3) product channel for the photodissociation of CH(3)SH at 204 nm was investigated using the sliced velocity map ion imaging technique with the detection of CH(3) products using state selective (2+1) resonance enhanced multiphoton ionization (REMPI). Images were measured for CH(3) formed in the ground and excited vibrational states (v(2) = 0, 1, and 2) of the umbrella mode from which the correlated SH vibrational state distributions were determined. The vibrational distribution of the SH fragment in the SH + CH(3) channel at 204 nm is clearly inverted and peaks at v = 1. The highly negative anisotropy parameter of the CH(3) (v(2) = 0, 1, and 2) products is indicative of a fast dissociation process for C-S bond cleavage. Two kinds of slower CH(3) products were also observed (one of which was partly vibrationally resolved) that are assigned to a two-step photodissociation processes, in which the first step is the production of the CH(3)S (X(2)E) radical via cleavage of the S-H bond in CH(3)SH, followed by probe laser photodissociation of nascent CH(3)S radicals yielding CH(3)(X(2)A(1), v(2) = 0-2) + S((3)P(j)/(1)D) products.

Product pair correlation in CH3OH photodissociation at 157 nm: the OH + CH3channel

The OH + CH(3) product channel for the photodissociation of CH(3)OH at 157 nm was investigated using the velocity map imaging technique with the detection of CH(3) radical products via (2+1) resonance-enhanced multiphoton ionization (REMPI). Images were measured for the CH(3) formed in the ground and excited states (v(2) = 0, 1, 2, and 3) of the umbrella vibrational mode and correlated OH vibrational state distributions were also determined. We find that the vibrational distribution of the OH fragment in the OH + CH(3) channel is clearly inverted. Anisotropic distributions for the CH(3) (v(2) = 0, 1, 2, and 3) products were also determined, which is indicative of a fast dissociation process for the C-O bond cleavage. A slower CH(3) product channel was also observed, that is assigned to a two-step photodissociation process, in which the first step is the production of a CH(3)O(X (2)E) radical via the cleavage of the O-H bond in CH(3)OH, followed by probe laser photodissociation of the nascent CH(3)O radicals yielding CH(3)(X (2)A(1), v = 0) products.

A velocity map ion-imaging study on C 2H 5SH and C 3H 7SH photodissociation at 243 nm

Photodissociation dynamics of C 2H 5SH, i-C 3H 7SH and n-C 3H 7SH at 243.1 nm were investigated using velocity map ion-imaging method. H-atom photolysis products were detected by a (2 + 1) resonance enhanced ionization scheme. Both the angular distribution and translational energy distribution of the H-atom elimination processes were determined from the ion images of the H-atom products. The experimental results indicate that the H-atom eliminations from these molecules are mainly direct and fast dissociation processes from a repulsive potential energy state. And a more statistical dissociation process that likely occurs on the ground state via internal conversion has also been observed. Dissociation energies of the S–H bonds are also derived from the H-atom product translational energy distributions.

Determination of Binding Constants by Equilibrium Titration with Circulating Sample in a Surface Plasmon Resonance Biosensor

A commercial surface plasmon resonance biosensor, BIACORE X, is employed as a detector in a closed loop of a small sample volume. The sample is continuously circulated by an external syringe pump over two sensor spots, one functionalized with immobilized binding sites to a soluble binding partner in the mobile phase and one serving as a reference surface. A binding isotherm for the interacting macromolecules can be obtained by a stepwise titration of the soluble reactant into the circulating loop, each step followed by observation of the signal increase until equilibrium is attained. Binding constants can be measured under conditions free of mass transport artifacts and without the requirement for regeneration of the immobilized binding sites. This procedure is similar to the stepwise titration procedure described for the cuvette-based sensor design (D. R. Hall and D. J. Winzor, 1997,Anal. Biochem.244, 152–160). In the presented configuration, the high baseline stability of the instrument combined with the availability of a reference surface for the detection of nonspecific binding permits refractive index changes upon addition of the aliquots to be measured, as well as accounting for temperature or instrumental drifts, and allows for a very long experimental time. This feature extends the applicability of equilibrium titration to systems with higher affinity or slower dissociation rate constants. Furthermore a solution competition titration is described that avoids artifacts from the immobilization procedure to provide a method for measurement of binding constants in solution. Kinetic information on the complex dissociation can also be obtained by combination of sample delivery via the external pump with the injection of competitor via the microfluidics of the biosensor. The rapid injection of high concentrations of competitor allows the observation of fast dissociation processes under conditions minimizing rebinding.

Femtosecond Transition-State Dynamics of Dissociating OCS on the Excited 1Σ+ Potential Energy Surface

Ultrafast photodissociation dynamics of OCS on the dissociative potential energy surface (PES) of the electronically excited 1Σ+ state was investigated by photofragment excitation (PHOFEX) spectroscopy and time-dependent wavepacket calculations. The high-resolution PHOFEX spectrum of the entire 1Σ+−1Σ+ transition (63 300−69 350 cm-1) in the vacuum ultraviolet (vacuum UV) region was measured under jet-cooled conditions by using a tunable vacuum UV laser as an excitation light source and by monitoring the fragment S(1S) atom. Due to sufficient vibrational and rotational cooling in a supersonic jet, a simple and distinct vibrational progression of the 1Σ+−1Σ+ band was recorded free from vibrational hot bands and the broadening by rotational structure. The autocorrelation function obtained from a Fourier transform of the PHOFEX spectra clearly exhibited recurrences with a period of 42 fs, corresponding to a period of the vibrational motion at the transition state along the direction perpendicular to the dissociation coordinate. This interpretation of the ultrafast motion in the transition-state region was supported by the wavepacket calculation on the ab initio PES obtained in the present study, which afforded a period of 48 fs for vibrational motion along the in-phase CO and CS stretching mode at the transition state on the ab initio PES. The distinct six peaks broadened due to the fast dissociation process were assigned to the transitions at the vTS = 0−5 levels, where vTS represents a vibrational quantum number for the Feshbach resonances in the in-phase stretching mode at the transition state. For the vTS = 0 peak, the narrowest width corresponding to the lifetime, τ(vTS), of τ(0) = 133 fs was obtained. It was found that the dissociation lifetime first becomes shorter for a larger vTS, i.e., τ(1) = 44 fs and τ(2) = 27 fs, and then, the peak width becomes narrower for the higher vibrational states above vTS = 2, with corresponding lifetimes of τ(3) = 47 fs and τ(4) = 44 fs. This deceleration of the dissociation rate for the larger vTS states was interpreted as a result of trapping of a wave function in the in-phase stretching or the CO stretching vibrational motion suppressing the motion along the dissociation coordinate. All six main peaks in the PHOFEX spectra exhibited a characteristic asymmetric profile, originating from the interference between the zero-order discrete states for the in-phase stretching vibration at the transition state and the zero-order continuum states corresponding with the motion along the dissociation coordinate. In the main progression of the PHOFEX spectra, the reversal of the asymmetry direction was also observed. This q-reversal phenomenon was ascribed to the characteristic shape of the excited 1Σ+ PES near the transition region which causes a phase shift in the wave functions in a narrow energy range.

Hydrogen-bonding interactions in gas-phase polyether/ammonium ion complexes

Hydrogen bonds are among the most important interactions involved in selective complexation in host—guest chemistry. In this study a variety of hydrogen-bonded crown ether/ammonium ion complexes are generated in the gas phase by association reactions between an amine substrate and a polyether, one of which is initially protonated, and stabilized by many collisions in the chemical ionization source of a triple quadrupole mass spectrometer or in a quadrupole ion trap. The nature of the hydrogen-bonding interactions of the ion complexes are evaluated by comparison of their collision-activated dissociation spectra. After collisional activation, those complexes that are weakly bound dissociate to form intact protonated polyether molecules and/or ammonium ions by simple cleavages of the hydrogen-bond association interactions. In contrast, those complexes strongly bound by multiple hydrogen bonds dissociate not only to the protonated polyether and/or ammonium ions but also by extensive covalent bond cleavage of the protonated ether skeleton. This latter type of dissociation behavior suggests that the polyether/ammonium ion complexes may be sufficiently strongly bound that surpassing the high barrier to decomposition results in formation of internally excited polyether molecules that may then undergo subsequent fragmentation by skeletal cleavages. Moreover, complexes involving multiple hydrogen bonds may have slower dissociation kinetics, allowing competition from fast dissociation processes that have substantial energy barriers.

Photoionization Decay of Core Excited Molecules

The excitation of a core electron into an antibonding empty valence orbital of a molecule leads to a quite intense resonance compared to Rydberg transitions in the photoabsorption spectrum. We show the direct experimental evidence of its repulsive character by photoelectron spectroscopy in the case of a d-shell excitation of halogen compounds such as HBr, CH3Br, HI and CH3I. The first step of the resonance decay is a very fast dissociation process followed by autoionization of the core excited fragment as a second step. Competition with usual molecular autoionization is found when the mass of the radical linked to the excited atom is increased.

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. Dissociation of internalized ligand from receptor occurs in two kinetically and thermally distinguishable compartments.

We have examined the rate of dissociation of internalized 125I-asialo-orosomucoid-receptor complexes in freshly isolated rat hepatocytes. Cell suspensions were washed with ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 0 degrees C to remove surface-bound ligand and then assessed for the retention of radioactive glycoprotein in the presence of digitonin, which permeabilized the cells and released the internal soluble contents. In cells which initially contained only surface-bound ligand, about 50% of the internalized ligand dissociated from receptor very rapidly (t1/2 less than or equal to 2.5 min, k greater than or equal to 0.28 min-1), at 37 degrees C, whereas the other 50% dissociated more slowly with apparent first order kinetics (t1/2 = 50 min, k = 0.014 min-1). This equal distribution of internalized ligand into two compartments, from which dissociation occurred with very different kinetics, did not depend on the extent of surface receptor occupancy and also occurred under non-steady state conditions of continuous exposure to ligand. Ligand entering both the rapid and slow dissociation compartments was eventually degraded with apparent first order kinetics (k = 0.0047 min-1), suggesting that the intracellular routing of ligand to lysosomes after dissociation from either compartment was via the same pathway. The fast and slow dissociation of receptor-ligand complexes were also distinguished by different temperature sensitivities; the slow dissociation process ceased below 18 degrees C, whereas the fast dissociation process still proceeded. The equal partition of internalized complexes into the two kinetic compartments did not change as a function of temperature but did change as cells continued to endocytose asialo-orosomucoid at 37 degrees C. As the internal receptor pool approached a steady state level of occupancy, there was an increase in the average time for receptor recycling and an increase in the fraction of incoming receptor-ligand complexes which dissociated rapidly (approximately 75%). In addition, under steady state conditions, the rate of the slow dissociation process increased (k = 0.026 min-1, t1/2 = 27 min).

Collision- and non-collision-induced predissociation in the appearance of S + and CS + ions from CS 2 under electron impact

Metastable transitions in CS + 2 leading to S + + CS and CS + + S under electron impact on CS 2 ae reported. The predissociation processes able to explain the occurrence of these metastable transitions are discussed. An interpretation of some fast dissociation processes is suggested.