The αβ T cell receptor (TCR) repertoire on mature T cells is selected in the thymus, but the basis for thymic selection of MHC-restricted TCRs from a randomly generated pre-selection repertoire is not known. Here we perform comparative repertoire sequence analyses of pre-selection and post-selection TCR from multiple MHC-sufficient and MHC-deficient mouse strains, and find that MHC-restricted and MHC-independent TCRs are primarily distinguished by features in their non-germline CDR3 regions, with many pre-selection CDR3 sequences not compatible with MHC-binding. Thymic selection of MHC-independent TCR is largely unconstrained, but the selection of MHC-specific TCR is restricted by both CDR3 length and specific amino acid usage. MHC-restriction disfavors TCR with CDR3 longer than 13 amino acids, limits positively charged and hydrophobic amino acids in CDR3β, and clonally deletes TCRs with cysteines in their CDR3 peptide-binding regions. Together, these MHC-imposed structural constraints form the basis to shape VDJ recombination sequences into MHC-restricted repertoires.

Xanthorhodopsin is a retinal protein, including carotenoid light harvesting antenna. femtosecond VIS to NIR study demonstrating efficient ultrafast light harvesting from carotenoid to retina is described. Dynamics and intermediates are discussed.

The lysis-lysogeny decision of bacteriophage lambda has been a paradigm for a developmental genetic network, which is composed of interlocked positive and negative feedback loops. This genetic network is capable of responding to environmental signals and to the number of infecting phages. An interplay between CI and Cro functions suggested a bistable switch model for the lysis-lysogeny decision. Here, we present a real-time picture of the execution of lytic and lysogenic pathways with unprecedented temporal resolution. We monitor, in vivo, both the level and function of the CII and Q gene regulators. These activators are cotranscribed yet control opposite developmental pathways. Conditions that favor the lysogenic response show severe delay and down-regulation of Q activity, in both CII-dependent and CII-independent ways. Whereas CII activity correlates with its protein level, Q shows a pronounced threshold before its function is observed. Our quantitative analyses suggest that by regulating CII and CIII, Cro plays a key role in the ability of the lambda genetic network to sense the difference between one and more than one phage particles infecting a cell. Thus, our results provide an improved framework to explain the longstanding puzzle of the decision process.

Well-oriented monolayers of bacteriorhodopsin(bR)-containing purple-membrane patches are prepared on solid substrates (see Figure). Green-light illumination completely converts wild-type bR to the blue-light-absorbing M state, even at high humidity and pH 7. The possibility of bR-based optoelectronic devices is significantly enhanced by systems comprised of the long-lived M state, thus underlying the importance of this work.

The all-trans to 13-cis photoisomerization of the retinal chromophore in bacteriorhodopsin (bR) acts as a strong steric and electronic perturbation for the protein environment, thereby driving the proton pump mechanism. Photoexcitation and torsional movement are expected to be paralleled by various intra- and intermolecular relaxation processes. According to femtosecond (fs) time-resolved optical absorption spectroscopy the electronic ground state is reached after about 500 fs (J) [1-4], which then relaxes on a 3 ps time-scale to the K-state with the chromophore in 13-cis configuration.

The special trimeric structure of bacteriorhodopsin (bR) in the purple membrane of Halobacterium salinarum, and especially, the still controversial question as to whether the three protein components are structurally and functionally identical, have been subject to considerable work. In the present work, the problem is approached by studying the reconstitution reaction of the bR apo-protein with all-trans retinal, paying special attention to the effects of the apo-protein/retinal (P:R) ratio. The basic observation is that at high P:R values, the reconstitution reaction proceeds via two distinct, fast and slow, pathways associated with two different pre-pigment precursors absorbing at 430 nm (P(430)) and 400 nm (P(400)), respectively. These two reactions, exhibiting 2:1 (P(430)/P(400)) amplitude ratios, are markedly affected by the P:R value. The principal feature is the acceleration of the P(400) --> bR transition at low P:R ratios. The data are interpreted in terms of a scheme in which the added retinal first occupies two protein retinal traps, R(1) and R(2), from which it is transferred to two spectroscopically distinct binding sites corresponding to the two pre-pigments, P(430) and P(400), respectively. Two noncovalently bound retinal molecules occupy two P(430) sites of the bR trimer, while one (P(400)) occupies the third. Binding is completed by generating the retinal-protein covalent bond. Analogous experiments were also carried out with an aromatic bR chromophore and with the D85N bR mutant. The accumulated data clearly point out the heterogeneity of the binding reaction intermediates, in which two are clearly distinct from the third. However, CD spectroscopy strongly suggests that even the two P(430) sites are not structurally identical. The heterogeneity of the P intermediates in the binding reaction can be accounted for, either by being induced by cooperativity or by an intrinsic heterogeneity that is already present in the apoprotein. The question as to whether the final reconstituted pigment, as well as native bR, are nonhomogeneous should be the subject of future studies.

Low frequency excited state vibrational coherences induced by impulsive photoexcitation in bacteriorhodopsin are detected via femtosecond pump–probe spectroscopy, and compared with similar data in retinal protonated Schiff bases of native and locked retinals. At delays above ∼100 fs a single vibration below 200 fs dominates the detected spectral modulations. Its frequency of ∼120 in retinal protonated Schiff base is virtually unchanged by locking the C13C14 bond in the trans or cis configurations, but is increased to 170 cm−1 within the protein environment. The implications of this result on the part played by the protein in directing the reactivity of the retinal within bacteriorhodopsin is discussed.

We propose a new scheme for constructing a single-beam dark optical trap that minimizes light-induced perturbations of the trapped atoms. The proposed scheme optimizes the trap depth for given trapping laser power and detuning by creating a light envelope with (a) an almost minimal surface area for a given volume and (b) the minimal wall thickness that is allowed by diffraction. The stiffness of the trap's walls, combined with the large detuning allowed by the efficient distribution of light intensity, yields a low spontaneous photon scattering rate for the trapped atoms. Our trap also optimizes the loading efficiency by maximizing the geometrical overlap between a magneto-optical trap and the dipole trap. We demonstrate this new scheme by generating the proposed light distribution of a single-beam dark trap with a trap depth that is similar to33 times larger than that of existing blue-detuned traps and similar to13 times larger than that of a red-detuned trap with the same diameter, detuning, and laser power. Trapped atoms are predicted to have a decoherence rate that is >200 times smaller than in existing single-beam dark traps and similar to1800 times smaller than in a red-detuned trap with the same diameter, depth, and laser power. (C) 2002 Optical Society of America.

Time-integrated fluorescence experiments on native bacteriorhodopsin and on its non-isomerizing form bR5.12 are reported. The experimental set-up was designed such as to observe emission exclusively from the excited state intermediate I-460. We obtain the first systematic investigation of the fluorescence spectra as a function of the excitation wavelength tuned throughout the entire absorption band of bR. An important finding is that the position of the fluorescence maximum does not show a systematic shift when the excitation wavelength is shortened. For excitation with high excess energy, we observe a broadening of the blue wing of the bR fluorescence, indicating incomplete vibrational energy relaxation on the time scale of the lifetime of I-460. Due to a much longer excited state lifetime, vibrational energy relaxation is more effective in bR5.12 and the fluorescence spectra are much less dependent on excitation wavelength. The results are placed in the general framework of thermalization between the retinal chromophore and the protein environment, and are compared with information obtained by femtosecond experiments.

Summary form only given. Blue-detuned optical traps confine neutral atoms mostly in the dark, therefore reducing the perturbations induced by the trapping light on the atoms. Recently, traps based on a single laser beam were demonstrated, providing greater experimental simplicity and enabling dynamical changes of the trap geometry and strength. However, all these traps have a large difference in the potential strength in the radial and axial directions, resulting in a very inefficient use of the available laser power, and a poor loading efficiency from the magneto-optical trap. In this work we present a novel optical scheme to generate an optimal trap, which maximizes the trap depth, and greatly reduces the light-induced perturbations to the atoms. This is achieved by surrounding a dark volume with a light envelope having (a) an almost minimal surface area for a given volume, (b) the minimal wall thickness allowed by diffraction, and (c) an almost constant wall height on every point on the envelope. The stiffness of the walls, combined with the large detuning allowed by the efficient distribution of light intensity, yield a very low photon scattering rate for the trapped atoms.

New information concerning the photochemical dynamics of bacteriorhodopsin (BR) is obtained by impulsively stimulating emission from the reactive fluorescent state. Depletion of the excited-state fluorescence leads to an equal reduction in production of later photoproducts. Accordingly, chromophores which are forced back to the ground state via emission do not continue on in the photocycle, conclusively demonstrating that the fluorescent state is a photocycle intermediate. The insensitivity of depletion dynamics to the "dump" pulse timing, throughout the fluorescent states lifetime, and the biological inactivity of the dumped population suggest that the fluorescent-state structure is constant, well-defined, and significantly different than that where crossing to the ground state takes place naturally. In conjunction with conclusions from comparing the photophysics of BR with those of synthetic analogues containing "locked" retinals, present results show that large-amplitude torsion around C13=C14 is required to go between the above structures.

We investigate the effects of curving trajectories by applying external force fields on a particle in a billiard. We investigate two special cases: a constant force field and a parabolic potential. These perturbations change the stability conditions and can lead to formation of elliptical orbits in otherwise hyperbolic billiards. We demonstrate these effects experimentally with ultra-cold atoms in atom-optic billiards.

The primary events in the photosynthetic retinal protein bacteriorhodopsin (bR) are reviewed in light of photophysical and photochemical experiments with artificial bR in which the native retinal polyene is replaced by a variety of chromophores. Focus is on retinals in which the “critical” C13=C14 bond is locked with respect to isomerization by a rigid ring structure. Other systems include retinal oxime and non-isomerizable dyes noncovalently residing in the binding site. The early photophysical events are analyzed in view of recent pump–probe experiments with sub-picosecond time resolution comparing the behavior of bR pigments with those of model protonated Schiff bases in solution. An additional approach is based on the light-induced cleavage of the protonated Schiff base bond that links retinal to the protein by reacting with hydroxylamine. Also described are EPR experiments monitoring reduction and oxidation reactions of a spin label covalently attached to various protein sites. It is concluded that in bR the initial relaxation out of the Franck–Condon (FC) state does not involve sub-stantial C13=C14 torsional motion and is considerably catalyzed by the protein matrix. Prior to the decay of the relaxed fluorescent state (FS or I state), the protein is activated via a mechanism that does not require double bond isomerization. Most plausibly, it is a result of charge delocalization in the excited state of the polyene (or other) chromophores. More generally, it is concluded that proteins and other macromolecules may undergo structural changes (that may affect their chemical reactivity) following optical excitation of an appropriately (covalently or non-covalently) bound chromophore. Possible relations between the light-induced changes due to charge delocalization, and those associated with C13=C14 isomerization (that are at the basis of the bR photocycle), are discussed. It is suggested that the two effects may couple at a certain stage of the photocycle, and it is the combination of the two that drives the cross-membrane proton pump mechanism.