Kinetics of DNA duplex formation: A-tracts versus AT-tracts.

The hybridisation and melting of DNA strands are critical steps in many biological processes, but still a deeper understanding of the kinetics is lacking. This is evident from the absence of a clear correlation between rate constants for duplex formation and the number of bases in the strand or the sequence. Here we have probed differences between formation times of A-tracts and AT-tracts by studying complementary model strands mainly comprised of adenine (A) and thymine (T) in stopped-flow (SF) experiments. These strands are relevant as DNA replication begins in regions with a large number of AT base pairs. Interpretation of our results is aided by secondary-structure modelling where both the fractions of the different types of structures and the number of paired bases in the lowest-energy ones are determined. The model is based on calculation of free energies using fixed values for enthalpies and entropies associated with base pairing and a stochastic sampling of the possible structures. We find that the strand length affects rates: the activation energy for the formation of short (16-base pairs) A-tracts is larger than that for longer ones (20-base pairs). Activation energies for the formation of AT-tracts are an order of magnitude larger, and larger for shorter strands than for long ones. These higher activation energies are in agreement with the fact that the fraction of unpaired bases in the constituent AT-tract strands is less than in those which comprise the A-tracts. That the pre-structures of the single strands significantly affect rates is also used to rationalise the results obtained for two pairs of complementary 12-mer strands that have the same bases but in a different sequence; we report here similar activation energies as reported earlier and that these are strongly sequence dependent. Finally, we demonstrate that SF can be coupled with the measurement of circular dichroism (CD) in the vacuum ultraviolet (VUV) region, taking advantage of a synchrotron radiation facility, and that CD is useful to probe geometrical structures in the VUV where the absorption by DNA is high. Though this work is preliminary, our initial results suggest that the strands align prior to the formation of base pairs.

Collisional electron transfer to photoexcited acceptor radical anions.

In this article, we show that photoexcitation of radical anions facilitates electron transfer from sodium atoms in femtosecond encounters. Thus, excitation of 7,7,8,8-tetracyano-p-quinodimethane (TCNQ) and fluorinated TCNQ (TCNQ-F(4)) anions to the second optically active state at 478 nm led to increases in the yields of dianions of about 20% and 10%, respectively. Photoexcitation with a nanosecond-long laser pulse was done a few microseconds before the ions entered the sodium collision cell so that none of the ions would be in any of the initially reached doublet-excited states. We suggest an explanation for the higher electron capture cross section based on the formation of long-lived quartet state anions. Excitation of TCNQ anions within the lowest-energy absorption band, where there are no accessible quartet states, led instead to a lower yield of dianions. There are at least three explanations for the lower dianion yields: (1) Depletion of the monoanion beam due to photodetachment after the absorption of minimum two photons; (2) Formation of short-lived vibrationally excited dianions that decay by electron autodetachment prior to identification; and (3) Lower electron capture cross sections of vibrationally excited monoanions. Similar losses in dianion signal can occur at 478 nm so the actual yield of dianions at this wavelength due to the population of quartet states is therefore greater than that observed. Our methodology devises a more efficient route for the production of molecular dianions, and at the same time it may provide information on long-lived electronic states.

Electron-capture induced dissociation of doubly charged dipeptides: on the neutral losses and N-Cα bond cleavages.

Electron capture by doubly charged peptide cations leads to neutral losses in addition to N-C(α) bond cleavages that give c and z fragments. In this work we discuss the influence of amino acid sequence on hydrogen versus ammonia loss and the propensity for subsequent partial side-chain cleavage after ammonia loss to give w fragment ions. Experiments were done on two series of doubly protonated dipeptides, [XK+2H](2+) and [XR+2H](2+), where X is one of the twenty common amino acid residues, excluding aspartic acid (D), and K and R are lysine and arginine, respectively. While it was previously established that NH(3) is lost exclusively from the N-terminal ammonium group and not from side-chain ammonium groups, we find here that ammonia can be lost from guanidinium radicals as well. The ratio between H loss and NH(3) loss reveals some information on internal ionic hydrogen bonds and peptide conformation since proton sharing between the N-terminal ammonium group and a basic side chain decreases the probability for NH(3) loss due to a lower recombination energy and as a result reduced capture probability. The abundance of w ions was found to correlate with the reaction energy for their formation; highest yield was found for CK and lowest for AK and HK. The survival rate of charge-reduced species was higher for XR than for XK, which is likely linked to the formation of long-lived C(α) radicals in the latter case. The probability for N-C(α) bond cleavage is smaller on average for XR than for XK which indicates that hydrogen transfer from the ε-ammonium radical to the amide group triggers some of the cleavages, or is a result of the different distances between the amide group and the charges in XR and XK. Finally, our data support the previous concept that charge partitioning between c and z fragments can be explained by competition between the two fragments for the proton.

Electron capture induced dissociation of dipeptide dications: where does the charge go?

Charge partitioning after electron capture induced dissociation of dipeptide dications is determined by proton mobility in the evanescent ion-molecule complex as the remaining proton has enough time to choose the fragment with the highest proton affinity.

Absorption in the Q-band region by isolated ferric heme+ and heme+(histidine) in vacuo.

Absorption by heme proteins is determined by the heme microenvironment that is often vacuumlike (hydrophobic pocket). Here we provide absorption spectra in the Q-band region of isolated ferric heme(+) and heme(+)(histidine) ions in vacuo to be used as references in protein biospectroscopy. Ions were photoexcited in an electrostatic storage ring and their decay monitored in time. Both ions display a triple band structure with maxima at 500, 518, and 530 nm. Previous attempts to study four-coordinate Fe(III)-heme(+) were hampered by the strong affinity of Fe(3+) for water and anions. Absorption at higher wavelengths is also measured, which is ascribed to charge-transfer transitions from the porphyrin to the iron. Finally, our data serve to benchmark theoretical calculations.

Laser pump-probe experiments on microsecond to millisecond timescales at an electrostatic ion storage ring: triplet-triplet absorption by protoporphyrin-IX anions.

Here we demonstrate that pump-probe experiments can be carried out on microsecond to millisecond timescales using an electrostatic ion storage ring. As a test case, we have chosen protoporhyrin IX anions that have lifetimes with respect to dissociation after photoexcitation on this time scale. Ions were photoexcited on one side of the ring with either 430- or 535-nm light (pump) and then allowed to take a certain number of revolutions before they were photoexcited by a second laser pulse (probe) with wavelengths between 650 and 950 nm. If ions were first excited by the pump, an increased yield of neutral products caused by the absorption of red light was measured in a microchannel plate detector located on the other side of the ring. This implies that it is possible to pick out ions that were photoexcited by the pump pulse and to spectroscopically characterize these ions. We report absorption spectra of 535 nm photoexcited porphyrin anions, with time delays of 0.19 and 0.57 ms between the pump and probe pulses, and find that absorption occurs over a broad region in the red.

UV photodissociation of protonated Gly-Trp and Trp-Gly dipeptides and their complexes with crown ether in an electrostatic ion storage ring.

Photodissociation of protonated GW, WG (G = glycine, W = tryptophan), and their complexes with 18-crown-6-ether (CE) was performed in an electrostatic ion storage ring using a tunable laser system. On the basis of lifetime measurements, action spectra were obtained from 210 to 360 nm. These reveal that whereas [GW + H](+) absorbs maximally at approximately 220 nm, the absorption maximum is <210 nm for [WG + H](+), which is in good accordance with density functional theory calculations that predict band maxima at 221 and 212 nm, respectively. This difference in absorption is ascribed to the ammonium group interacting with the indole ring in the case of GW, thereby lowering the energy of the excited state more than that of the ground state. A broad band at higher wavelengths is observed for WG but not for GW, which again may be linked to differences in conformational structures between the two ions. Absorption spectra for the two CE tagged ions are very similar to each other: The high-energy band is now <210 nm for both peptide ions, and they display an absorption band with a maximum at 270 nm. The crown ether targets the ammonium protons, preventing an interaction between ammonium and indole, and the photophysics of the two complexes is therefore similar. The complexes have significantly longer lifetimes with respect to dissociation than the bare ions. Finally, we report product ion mass spectra at two different excitation wavelengths, 210 and 270 nm. There are significant differences between the two peptides, and the crown ether enhances certain channels, such as the loss of CO + H(2)O and the tryptophan side chain.

On the hydrogen loss from protonated nucleobases after electronic excitation or collisional electron capture.

In this work, we have subjected protonated nucleobases MH(+) (M = guanine, adenine, thymine, uracil and cytosine) to a range of experiments that involve high-energy (50 keV) collision induced dissociation and electron capture induced dissociation. In the latter case, both neutralisation reionisation and charge reversal were done. For the collision induced dissociation experiments, the ions interacted with O(2). In neutral reionisation, caesium atoms were used as the target gas and the protonated nucleobases captured electrons to give neutrals. These were reionised to cations a microsecond later in collisions with O(2). In choosing Cs as the target gas, we have assured that the first electron transfer process is favourable (by about 0.1-0.8 eV depending on the base). In the case of protonated adenine, charge reversal experiments (two Cs collisions) were also carried out, with the results corroborating those from the neutralisation reionisation experiments. We find that while collisional excitation of protonated nucleobases in O(2) may lead to hydrogen loss with limited probabilities, this channel becomes dominant for electron capture events. Indeed, when sampling reionised neutrals on a microsecond timescale, we see that the ratio between MH(+) and M(+) is 0.2-0.4 when one electron is captured from Cs. There are differences in these ratios between the bases but no obvious correlation with recombination energies was found.

Tagging of protonated Ala-Tyr and Tyr-Ala by crown ether prevents direct hydrogen loss and proton mobility after photoexcitation: importance for gas-phase absorption spectra, dissociation lifetimes, and channels.

Photodissociation of protonated Tyr, Ala-Tyr, Tyr-Ala (Ala = alanine, Tyr = tyrosine), and their complexes with 18-crown-6-ether (CE) was performed in an electrostatic ion storage ring using a tunable laser system. While the three bare ions all absorb strongly at 222 nm, absorption at higher wavelengths was barely visible from sampling the neutrals formed in delayed dissociation. A band at 270 nm was introduced, however, as a consequence of CE attachment to the bare ions. To understand the difference between bare ions and complexes, electronically excited states are considered: The initially reached pipi* state on phenol couples with the dissociative pisigma* state on ammonium, which leads to direct hydrogen loss. Cold radical cations are formed that at high wavelengths do not have enough energy for further dissociation. Excitation within the 222-nm band on the other hand leads to delayed dissociation of stored radical cations that is monitored in the present setup. The pisigma* state moves out of the spectral region upon CE attachment, and instead statistical dissociation is sampled on the microsecond to millisecond time scale at all wavelengths. Our data demonstrate the strength of using supramolecular complexes for action spectroscopy experiments to prevent erroneous spectra as a result of undesired dissociation (H loss) from electronically excited states. The gas-phase absorption spectra firmly establish the perturbations of the phenol electronic structure by a water solvent: The 270-nm band red shifts by approximately 5 nm, whereas the 222-nm band changes by approximately 3 nm. Both transitions occur in the phenol group. These results may be useful for protein dynamics experiments that rely on electronic excitations. Product ion mass spectra of [Tyr + H]+, [Ala-Tyr + H]+, [Tyr-Ala + H]+, [Ala-Tyr + H]+(CE), and [Tyr-Ala + H]+(CE) significantly depend on the excitation wavelength from 210 to 310 nm and on whether the ionizing proton is mobile or not.

Photodissociation of isolated ferric heme and heme-His cations in an electrostatic ion storage ring.

Photodissociation of isolated Fe(III)-heme(+) and Fe(III)-heme(+)(His) ions in the gas phase has been investigated using an electrostatic storage ring. The experiment provides three pieces of information, namely fragmentation channels, dissociation times, and absorption spectra. After photoexcitation with either 390 or 532 nm light, we find that the fragmentation takes place on a microsecond to millisecond time scale, and the channels are CH(2)COOH loss (beta-cleavage reaction) and histidine loss from Fe(III)-heme(+) and Fe(III)-heme(+)(His), respectively. These channels were also observed by means of collision-induced dissociation. Significant information on the nonradiative processes that occur after photoexcitation was revealed from the decay spectra. At early times (first two to three milliseconds), the decay of the photoexcited ions is well-described by a statistical model based on an Arrhenius-type expression for the rate constant. The activation energy and preexponential factor are 1.9 +/- 0.2 eV and 1 x 10(17) to 1 x 10(21) s(-1) for heme(+) and 1.4 +/- 0.2 eV and 1 x 10(16) to 1 x 10(19) s(-1) for heme(+)(His). Decay on a longer time scale was also observed and is ascribed to the population of lower-lying states with higher spin multiplicity because intersystem crossing back to the electronic ground-state is a bottleneck for the dissociation. The measurements give lifetimes for these lower-lying states of about 10 ms after 390 nm excitation and we estimate the probability of spin flip to be 0.3 and 0.8 for heme(+) and heme(+)(His), respectively.

Gas phase fragmentation of protonated betaine and its clusters.

Betaine [(CH(3))(3)N(+)CH(2)COO(-)] is a methylated version of glycine and is a zwitterion in its neutral form. In this work, we have subjected protonated betaine, (+)(CH(3))(3)NCH(2)COOH, to a range of fragmentation experiments which involve vibrational excitation, electronic excitation and electron capture. Low-energy (eV) collisions in combination with deuterium labelling reveal that the lowest energy dissociation pathway is the formation of N(CH(3))(3)(+) and CH(2)COOH. The dominant channel after 50 keV collisions with molecular oxygen is the same as that after low-energy collisions; however, more fragmentation is seen which is most likely due to electronic excitation of the ions in the collision processes. Subsequent dissociation of the radical N(CH(3))(3)(+) was observed in agreement with the electron ionisation spectrum of N(CH(3))(3). Electron-induced dissociation by 22 eV electrons produced similar fragments to those formed after high-energy collision-induced dissociation. With caesium atoms as the target gas, protonated betaine captured electrons to give neutrals. These were reionised to cations a microsecond later in collisions with O(2). The dominant dissociation channel of the betaine radical, [(CH(3))(3)NCH(2)COOH] , involves formation of N(CH(3))(3) and CH(2)COOH, as revealed from the presence of N(CH(3))(3)(+) radical cations. This channel is associated with a kinetic energy release of 0.1-0.2 eV. The CH(2)COOH radical is unstable to dissociation into CH(3) and CO(2) but in charge reversal experiments (two Cs collisions), CH(2)[double bond, length as m-dash]C(OH)O(-) anions were formed due to the short time between the collisions (nanoseconds). Density functional theory calculations support the spectral interpretations. Collision-induced dissociation of protonated betaine clusters resulted dominantly in loss of neutral betaines.

Photodissociation of protonated tryptophan and alteration of dissociation pathways by complexation with crown ether.

The behavior of protonated tryptophan (TrpH(+)) and its complex with 18-crown-6-ether (CE) after photoexcitation has been explored based on measurements of dissociation lifetimes, fragmentation channels, and absorption spectra using an electrostatic ion storage ring. A recent implementation of pulsed power supplies for the ring elements with microsecond response times allows us to identify the daughter ion fragment masses and to disentangle fragmentation that occurs from excited states immediately after photoexcitation from that occurring on a longer time scale of several microseconds to milliseconds. We find that attachment of crown ether significantly alters the dissociation channels since it renders the pisigma(*)(NH(3)) state inaccessible and hence prevents the N-H bond breakage which is an important fragmentation channel of TrpH(+). As a result, on a long time scale (>10 micros), photoexcited TrpH(+)(CE) decays exponentially whereas TrpH(+) displays a power-law decay. The only ions remaining in the latter case are Trp(+) radical cations with a broad internal energy distribution caused by the departing hydrogen. Large changes in the fragment branching ratios as functions of excitation wavelength between 210 and 290 nm were found for both TrpH(+) and TrpH(+)(CE).

A Soret marker band for four-coordinate ferric heme proteins from absorption spectra of isolated Fe(III)-Heme+ and Fe(III)-Heme+(His) ions in vacuo.

In this work, we report the absorption spectra in the Soret band region of isolated Fe(III)-heme+ and Fe(III)-heme+(His) ions in vacuo from action spectroscopy. Fe(III)-heme+ refers to iron(III) coordinated by the dianion of protoporphyrin IX. We find that the absorption of the five-coordinate complex is similar to that of pentacoordinate metmyoglobin variants with hydrophobic binding pockets except for an overall blueshift of about 16 nm. In the case of four-coordinate iron(III), the Soret band is similar to that of five-coordinate iron(III) but much narrower. These spectra serve as a benchmark for theoretical modeling and also serve to identify the coordination state of ferric heme proteins. To our knowledge this is the first unequivocal spectroscopic characterization of isolated 4c ferric heme in the gas phase.