Femtosecond observation of benzyne intermediates in a molecular beam: Bergman rearrangement in the isolated molecule

In this communication, we report our femtosecond real-time observation of the dynamics for the three didehydrobenzene molecules (p-, m-, and o-benzyne) generated from 1,4-, 1,3-, and 1,2-dibromobenzene, respectively, in a molecular beam, by using femtosecond time-resolved mass spectrometry. The time required for the first and the second C-Br bond breakage is less than 100 fs; the benzyne molecules are produced within 100 fs and then decay with a lifetime of 400 ps or more. Density functional theory and high-level ab initio calculations are also reported herein to elucidate the energetics along the reaction path. We discuss the dynamics and possible reaction mechanisms for the disappearance of benzyne intermediates. Our effort focuses on the isolated molecule dynamics of the three isomers on the femtosecond time scale.

Factors controlling the competition among rotational and vibrational energy transfer channels in glyoxal

The state-to-state transfer of rotational and vibrational energy has been studied for S1 glyoxal (CHOCHO) in collisions with D2, N2, CO and C2H4 using crossed molecular beams. A laser is used to pump glyoxal seeded in He to its S1 zero point level with zero angular momentum about its top axis (K′ = 0). The inelastic scattering to each of at least 26 S1 glyoxal rotational and rovibrational levels is monitored by dispersed S1–S0 fluorescence. Various collision partners are chosen to investigate the relative influences of reduced mass and the collision pair interaction potential on the competition among the energy transfer channels. When the data are combined with that obtained previously from other collision partners whose masses range from 2 to 84 amu, it is seen that the channel competition is controlled primarily by the kinematics of the collisional interaction. Variations in the intermolecular potential play strictly a secondary role.

Femtosecond cluster studies of the solvated 7-azaindole excited state double-proton transfer

Presented here are femtosecond pump-probe studies on the water-solvated 7-azaindole dimer, a model DNA base pair. In particular, studies are presented that further elucidate the nature of the reactive and nonreactive dimers and also provide new insights establishing that the excited state double-proton transfer in the dimer occurs in a stepwise rather than a concerted manner. A major question addressed is whether the incorporation of a water molecule with the dimer results in the formation of species that are unable to undergo excited state double-proton transfer, as suggested by a recent study reported in the literature [Nakajima, A., Hirano, M., Hasumi, R., Kaya, K., Watanabe, H., Carter, C. C., Williamson, J. M. & Miller, T. (1997) J. Phys. Chem. 101, 392–398]. In contrast to this earlier work, our present findings reveal that both reactive and nonreactive dimers can coexist in the molecular beam under the same experimental conditions and definitively show that the clustering of water does not induce the formation of the nonreactive dimer. Rather, when present with a species already determined to be a nonreactive dimer, the addition of water can actually facilitate the occurrence of the proton transfer reaction. Furthermore, on attaining a critical hydration number, the data for the nonreactive dimer suggest a solvation-induced conformational structure change leading to proton transfer on the photoexcited half of the 7-azaindole dimer.

Even-odd alternation in mass spectrum of thymine and uracil clusters: Evidence of intracluster photodimerization

Multiphoton ionization of thymine and uracil clusters generated by a supersonic molecular beam gave rise to a remarkable alternation of mass spectral intensities between even- and odd-numbered clusters. Such alternation was observed in clusters of up to 30 molecules. Excitation to the two lowest electronically excited states seemed to be a strong prerequisite. In view of the well known photodimerization reaction of thymine and uracil in the bulk phase, it is proposed that such alternation in the mass spectral intensity resulted from formation of photodimer units within the cluster on intense UV irradiation. Several analogues of thymine with no known propensity for photodimerization in the bulk phase did not exhibit any sign of such alternation in the cluster mass spectrum. The intrinsic UV window for photodimerization, and hence photoinduced mammalian mutagenesis, was estimated to be approximately 210–280 nm, significantly narrower than the previously reported bulk values of 150–300 nm.

Reciprocal secretion of proteins by the bacterial type III machines of plant and animal pathogens suggests universal recognition of mRNA targeting signals

Bacterial pathogens of both animals and plants use type III secretion machines to inject virulence proteins into host cells. Although many components of the secretion machinery are conserved among different bacterial species, the substrates for their type III pathways are not. The Yersinia type III machinery recognizes some secretion substrates via a signal that is encoded within the first 15 codons of yop mRNA. These signals can be altered by frameshift mutations without affecting secretion of the encoded polypeptides, suggesting a mechanism whereby translation of yop mRNA is coupled to the translocation of newly synthesized polypeptide. We report that the type III machinery of Erwinia chrysanthemi cloned in Escherichia coli recognizes the secretion signals of yopE and yopQ. Pseudomonas syringae AvrB and AvrPto, two proteins exported by the recombinant Erwinia machine, can also be secreted by the Yersinia type III pathway. Mapping AvrPto sequences sufficient for the secretion of reporter fusions in Yersinia revealed the presence of an mRNA secretion signal. We propose that 11 conserved components of type III secretion machines may recognize signals that couple mRNA translation to polypeptide secretion.

Structural activity of a cloned potassium channel (ROMK1) monitored with the atomic force microscope: The “molecular-sandwich” technique

The atomic force microscope (AFM) was used to continuously follow height changes of individual protein molecules exposed to physiological stimuli. A AFM tip was coated with ROMK1 (a cloned renal epithelial potassium channel known to be highly pH sensitive) and lowered onto atomically flat mica surface until the protein was sandwiched between AFM tip and mica. Because the AFM tip was an integral part of a highly flexible cantilever, any structural alterations of the sandwiched molecule were transmitted to the cantilever. This resulted in a distortion of the cantilever that was monitored by means of a laser beam. With this system it was possible to resolve vertical height changes in the ROMK1 protein of ≥0.2 nm (approximately 5% of the molecule’s height) with a time resolution of ≥1 msec. When bathed in electrolyte solution that contained the catalytic subunit of protein kinase A and 0.1 mM ATP (conditions that activate the native ion channel), we found stochastically occurring height fluctuations in the ROMK1 molecule. These changes in height were pH-dependent, being greatest at pH 7.6, and lowering the pH (either by titration or by the application of CO2) reduced their magnitude. The data show that overall changes in shape of proteins occur stochastically and increase in size and frequency when the proteins are active. This AFM “molecular-sandwich” technique, called MOST, measures structural activity of proteins in real time and could prove useful for studies on the relationship between structure and function of proteins at the molecular level.

Bond orientational order, molecular motion, and free energy of high-density DNA mesophases.

By equilibrating condensed DNA arrays against reservoirs of known osmotic stress and examining them with several structural probes, it has been possible to achieve a detailed thermodynamic and structural characterization of the change between two distinct regions on the liquid-crystalline phase diagram: (i) a higher density hexagonally packed region with long-range bond orientational order in the plane perpendicular to the average molecular direction and (ii) a lower density cholesteric region with fluid-like positional order. X-ray scattering on highly ordered DNA arrays at high density and with the helical axis oriented parallel to the incoming beam showed a sixfold azimuthal modulation of the first-order diffraction peak that reflects the macroscopic bond-orientational order. Transition to the less-dense cholesteric phase through osmotically controlled swelling shows the loss of this bond orientational order, which had been expected from the change in optical birefringence patterns and which is consistent with a rapid onset of molecular positional disorder. This change in order was previously inferred from intermolecular force measurements and is now confirmed by 31P NMR. Controlled reversible swelling and compaction under osmotic stress, spanning a range of densities between approximately 120 mg/ml to approximately 600 mg/ml, allow measurement of the free-energy changes throughout each phase and at the phase transition, essential information for theories of liquid-crystalline states.

Molecular basis of human mitochondrial very-long-chain acyl-CoA dehydrogenase deficiency causing cardiomyopathy and sudden death in childhood.

beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the beta-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Very-long-chain acyl-CoA dehydrogenase [VLCAD; very-long-chain-acyl-CoA:(acceptor) 2,3-oxidoreductase, EC 1.3.99.13] catalyzes the first step in beta-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1-->a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C1837-->T, changing the arginine at residue 613 to tryptophan on one allele and a single base deletion at the intron-exon 6 boundary as the second mutation. This initial delineation of human mutations in VLCAD suggests that VLCAD deficiency reduces myocardial fatty acid beta-oxidation and energy production and is associated with cardiomyopathy and sudden death in childhood.