Several closely related third-order nonlinear time-resolved spectroscopic techniques, pump/probe transient absorption, transient grating, and three pulse stimulated photon echo peak shift measurements, are investigated theoretically and experimentally. It is shown in detail, through the consideration of response functions and numerical simulations including both finite pulse durations and detuning from exact resonance, how the solvation dynamics are manifested in these third-order nonlinear time-resolved spectroscopies. It is shown that the three pulse stimulated photon echo peak shift measurement and the transient grating measurement can give accurate dynamical information, whereas transient absorption may not be a reliable technique for a study of solvation dynamics in some cases. The contribution of very slow or static (inhomogeneous) components to the dynamics, however, can only be obtained from the three pulse echo peak shift measurements. Comprehensive experimental measurements are presented to illustrate and corroborate the calculations. We show that it is possible to separate the intramolecular vibrational and solvent contributions to the dephasing (or optical lineshape). Furthermore it is shown that the solvation of polar solutes in polar protic solvents has rather universal characteristics. The initial ultrafast process, usually identified as an inertial response of solvent molecules, occurs on a ∼100 fs time scale, and is essentially identical in methanol, ethanol, and butanol. The amplitude of this ultrafast component does, however, decrease with increasing alcohol size in 1-alkanols. The diffusive (≳0.5 ps) regime of the solvation process shows a strong solvent dependence, and may be described satisfactorily by dielectric relaxation theories.
Thymus-derived lymphocytes protect mammalian hosts against virus-or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric ␣, CD3⑀␥, CD3⑀␦, and CD3 subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3⑀ lobe that they approach diagonally, adjacent to the lever-like C FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3⑀ and CD3␥ in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force (ϳ50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands. The T cell receptor (TCR)3 is a multimeric transmembrane complex composed of a disulfide-linked antigen binding clonotypic heterodimer (␣ or ␥␦) in non-covalent association with the signal-transducing CD3 subunits (CD3⑀␥, CD3⑀␦, and CD3) (reviewed in Ref. 1). TCR signaling via CD3 dimers evokes T cell lineage commitment and repertoire selection during development, maintains the peripheral T cell pool, and further differentiates naïve T cells into effector or memory cell populations upon immune stimulation (2-5). The interaction between an Fab-like ␣ TCR heterodimer and an antigenic peptide bound to a major histocompatibility complex molecule (pMHC) initiates a cascade of downstream signaling events via the immunoreceptor tyrosine-based activation motif elements in the cytoplasmic tails of the associated CD3 subunits (6 -9). The length of these CD3 cytoplasmic tails is substantial, relative to those of the TCR ␣ and  chains (6, 7).How recognition of pMHC by a weakly interacting (ϳ1-100 M K d ) clonotypic heterodimer on the T cell surface evokes intracellular signaling via the adjacent CD3 components remains undefined (1). Solution structures of CD3⑀␥ and CD3⑀␦ heterodimers reveal a unique side-to-side hydrophobic interface with conjoined -sheets involving the G-strands of the two Ig-like ectodomains of the pair (10, 11). The squat and rigid CD3 connecting segments contrast sharply with the long and flexible TCR ␣ and  connecting peptides linking their respective constant domains to the transmembrane segments.To investigate the basis of signal transduction involving the ectodomain components within the TCR membrane complex,...
Photon echo spectroscopy is used to study the mechanisms of solvation dynamics in protein environments at room temperature. Ultrafast and additional multi-exponential long time scales are observed in the three-pulse photon echo peak shift data of the fluorescein dye eosin bound to lysozyme in aqueous solution. The dynamics of the solvated lysozyme are characterized with dielectric continuum models that integrate dielectric data for water with that for lysozyme. By comparing our data with previous results for eosin in water [Lang, M. J.; Jordanides, X. J.; Song, X.; Fleming, G. R. J. Chem. Phys. 1999, 110, 5584], we find that the total coupling of the electronic transition frequency of eosin to the nuclear motions of the aqueous lysozyme solution is smaller than in the aqueous solution. On an ultrafast time scale, solvation appears to be dominated by the surrounding water and not by the ultrafast internal motions of lysozyme. However, over long time scales, lysozyme does contribute significantly, either directly through motions of polar side chains or indirectly through reorientation of the water "bound" to the surface of the protein.
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