Protein dynamics observed by tunable mid-IR quantum cascade lasers across the time range from 10 ns to 1 s

We have developed a spectrometer based on tunable quantum cascade lasers (QCLs) for recording time-resolved absorption spectra of proteins in the mid-infrared range. We illustrate its performance by recording time-resolved difference spectra of bacteriorhodopsin in the carboxylic range (1800–1700 cm− 1) and on the CO rebinding reaction of myoglobin (1960–1840 cm− 1), at a spectral resolution of 1 cm− 1. The spectrometric setup covers the time range from 4 ns to nearly a second with a response time of 10–15 ns. Absorption changes as low as 1 × 10− 4 are detected in single-shot experiments at t > 1 μs, and of 5 × 10− 6 in kinetics obtained after averaging 100 shots. While previous time-resolved IR experiments have mostly been conducted on hydrated films of proteins, we demonstrate here that the brilliance of tunable quantum cascade lasers is superior to perform ns time-resolved experiments even in aqueous solution (H2O)

Photoexcitation of the P4480 state induces a secondary photocycle that potentially desensitizes channelrhodopsin-2

Channelrhodopsins (ChRs) are light-gated cation channels. In spite of their wide use to activate neurons with light, the photocurrents of ChRs rapidly decay in intensity under both continuous illumination and fast trains of light pulses, broadly referred to as desensitization. This undesirable phenomenon has been explained by two interconnected photocycles, each of them containing a nonconductive dark state (D1 and D2) and a conductive state (O1 and O2). While the D1 and O1 states correspond to the dark-state and P3520 intermediate of the primary all-trans photocycle of ChR2, the molecular identity of D2 and O2 remains unclear. We show that P4480, the last intermediate of the all-trans photocycle, is photoactive. Its photocycle, characterized by time-resolved UV/vis spectroscopy, contains a red-shifted intermediate, I3530. Our results indicate that the D2 and O2 states correspond to the P4480 and I3530 intermediates, connecting desensitization of ChR2 with the photochemical properties of the P4480 intermediate

Protein conformational changes and protonation dynamics probed by a single shot using quantum-cascade-laser-based IR spectroscopy

Mid-IR spectroscopy is a powerful and label-free technique to investigate protein reactions. In this study, we use quantum-cascade-laser-based dual-comb spectroscopy to probe protein conformational changes and protonation events by a single-shot experiment. By using a well-characterized membrane protein, bacteriorhodopsin, we provide a comparison between dual-comb spectroscopy and our homebuilt tunable quantum cascade laser (QCL)-based scanning spectrometer as tools to monitor irreversible reactions with high time resolution. In conclusion, QCL-based infrared spectroscopy is demonstrated to be feasible for tracing functionally relevant protein structural changes and proton translocations by single-shot experiments. Thus, we envisage a bright future for applications of this technology for monitoring the kinetics of irreversible reactions as in (bio-)chemical transformations

Defective Suppressor Function of Human CD4+ CD25+ Regulatory T Cells in Autoimmune Polyglandular Syndrome Type II

In autoimmune polyglandular syndromes (APS), several organ-specific autoimmune diseases are clustered. Although APS type I is caused by loss of central tolerance, the etiology of APS type II (APS-II) is currently unknown. However, in several murine models, depletion of CD4+ CD25+ regulatory T cells (Tregs) causes a syndrome resembling human APS-II with multiple endocrinopathies. Therefore, we hypothesized that loss of active suppression in the periphery could be a hallmark of this syndrome. Tregs from peripheral blood of APS-II, control patients with single autoimmune endocrinopathies, and normal healthy donors showed no differences in quantity (except for patients with isolated autoimmune diseases), in functionally important surface markers, or in apoptosis induced by growth factor withdrawal. Strikingly, APS-II Tregs were defective in their suppressive capacity. The defect was persistent and not due to responder cell resistance. These data provide novel insights into the pathogenesis of APS-II and possibly human autoimmunity in general

First Order Static Excitation Potential: Scheme for Excitation Energies and Transition Moments

We present an approximation scheme for the calculation of the principal excitation energies and transition moments of finite many-body systems. The scheme is derived from a first order approximation to the self energy of a recently proposed extended particle-hole Green's function. A hermitian eigenvalue problem is encountered of the same size as the well-known Random Phase Approximation (RPA). We find that it yields a size consistent description of the excitation properties and removes an inconsistent treatment of the ground state correlation by the RPA. By presenting a hermitian eigenvalue problem the new scheme avoids the instabilities of the RPA and should be well suited for large scale numerical calculations. These and additional properties of the new approximation scheme are illuminated by a very simple exactly solvable model.Comment: 15 pages revtex, 1 eps figure included, corrections in Eq. (A1) and Sec. II

The nuclear envelope protein Nesprin-2 has roles in cell proliferation and differentiation during wound healing

Nesprin-2, a type II transmembrane protein of the nuclear envelope, is a component of the LINC complex that connects the nuclear lamina with the actin cytoskeleton. To elucidate its physiological role we studied wound healing in Nesprin-2 Giant deficient mice and found that a loss of the protein affected wound healing particularly at later stages during fibroblast differentiation and keratinocyte proliferation leading to delayed wound closure. We identified altered expression and localization of transcription factors as one of the underlying mechanisms. Furthermore, the actin cytoskeleton which surrounds the nucleus was altered and keratinocyte migration was slowed down and focal adhesion formation enhanced. We also uncovered a new activity of Nesprin-2. When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA. Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation. Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing

The dynamical Green's function and an exact optical potential for electron-molecule scattering including nuclear dynamics

We derive a rigorous optical potential for electron-molecule scattering including the effects of nuclear dynamics by extending the common many-body Green's function approach to optical potentials beyond the fixed-nuclei limit for molecular targets. Our formalism treats the projectile electron and the nuclear motion of the target molecule on the same footing whereby the dynamical optical potential rigorously accounts for the complex many-body nature of the scattering target. One central result of the present work is that the common fixed-nuclei optical potential is a valid adiabatic approximation to the dynamical optical potential even when projectile and nuclear motion are (nonadiabatically) coupled as long as the scattering energy is well below the electronic excitation thresholds of the target. For extremely low projectile velocities, however, when the cross sections are most sensitive to the scattering potential, we expect the influences of the nuclear dynamics on the optical potential to become relevant. For these cases, a systematic way to improve the adiabatic approximation to the dynamical optical potential is presented that yields non-local operators with respect to the nuclear coordinates.Comment: 22 pages, no figures, accepted for publ., Phys. Rev.