Sequential conformational transitions and α-helical supercoiling regulate a sensor histidine kinase

Sensor histidine kinases are central to sensing in bacteria and in plants. They usually contain sensor, linker, and kinase modules and the structure of many of these components is known. However, it is unclear how the kinase module is structurally regulated. Here, we use nano- to millisecond time- resolved X-ray scattering to visualize the solution structural changes that occur when the light-sensitive model histidine kinase YF1 is activated by blue light. We find that the coiled coil linker and the attached histidine kinase domains undergo a left handed rotation within microseconds. In a much slower second step, the kinase domains rearrange internally. This structural mechanism presents a template for signal transduction in sensor histidine kinases

Communication: Nanosecond folding dynamics of an alpha helix: Time-dependent 2D-IR cross peaks observed using polarization-sensitive dispersed pump-probe spectroscopy

We present a simple method to measure the dynamics of cross peaks in time-resolved two-dimensional vibrational spectroscopy. By combining suitably weighted dispersed pump-probe spectra, we eliminate the diagonal contribution to the 2D-IR response, so that the dispersed pump-probe signal contains the projection of only the cross peaks onto one of the axes of the 2D-IR spectrum. We apply the method to investigate the folding dynamics of an alpha-helical peptide in a temperature-jump experiment and find characteristic folding and unfolding time constants of 260 ± 30 and 580 ± 70 ns at 298 K

Folding of a Zinc-Finger ββα-Motif Investigated Using Two-Dimensional and Time-Resolved Vibrational Spectroscopy

Small proteins provide good model systems for studying the fundamental forces that control protein folding. Here, we investigate the folding dynamics of the 28-residue zinc-finger mutant FSD-1, which is designed to form a metal-independent folded ββα-motif, and which provides a testing ground for proteins containing a mixed α/β fold. Although the folding of FSD-1 has been actively studied, the folding mechanism remains largely unclear. In particular, it is unclear in what stage of folding the α-helix is formed. To address this issue we investigate the folding mechanism of FSD-1 using a combination of temperature-dependent UV circular dichroism (UV-CD), Fourier transform infrared (FTIR) spectroscopy, two-dimensional infrared (2D-IR) spectroscopy, and temperature-jump (<i>T</i>-jump) transient-IR spectroscopy. Our UV-CD and FTIR data show different thermal melting transitions, indicating multistate folding behavior. Temperature-dependent 2D-IR spectra indicate that the α-helix is the most stable structural element of FSD-1. To investigate the folding/unfolding re-equilibration dynamics of FSD-1, the conformational changes induced by a nanosecond <i>T</i>-jump are probed with transient-IR and transient dispersed-pump–probe (DPP) IR spectroscopy. We observe biexponential <i>T</i>-jump relaxation kinetics (with time constants of 80 ± 13 ns and 1300 ± 100 ns at 322 K), confirming that the folding involves an intermediate state. The IR and dispersed-pump–probe IR spectra associated with the two kinetic components suggest that the folding of FSD-1 involves early formation of the α-helix, followed by the formation of the β-hairpin and hydrophobic contacts

Structure and dynamics of water in nonionic reverse micelles: A combined time-resolved infrared and small angle x-ray scattering study

We study the structure and reorientation dynamics of nanometer-sized water droplets inside nonionic reverse micelles (water/Igepal-CO-520/cyclohexane) with time-resolved mid-infrared pump-probe spectroscopy and small angle x-ray scattering. In the time-resolved experiments, we probe the vibrational and orientational dynamics of the O-D bonds of dilute HDO:H(2)O mixtures in Igepal reverse micelles as a function of temperature and micelle size. We find that even small micelles contain a large fraction of water that reorients at the same rate as water in the bulk, which indicates that the polyethylene oxide chains of the surfactant do not penetrate into the water volume. We also observe that the confinement affects the reorientation dynamics of only the first hydration layer. From the temperature dependent surface-water dynamics, we estimate an activation enthalpy for reorientation of 45 ± 9 kJ mol(-1) (11 ± 2 kcal mol(-1)), which is close to the activation energy of the reorientation of water molecules in ice

Water lubricates hydrogen-bonded molecular machines

The mechanical behaviour of molecular machines differs greatly from that of their macroscopic counterparts. This applies particularly when considering concepts such as friction and lubrication, which are key to optimizing the operation of macroscopic machinery. Here, using time-resolved vibrational spectroscopy and NMR-lineshape analysis, we show that for molecular machinery consisting of hydrogen-bonded components the relative motion of the components is accelerated strongly by adding small amounts of water. The translation of a macrocycle along a thread and the rotation of a molecular wheel around an axle both accelerate significantly on the addition of water, whereas other protic liquids have much weaker or opposite effects. We tentatively assign the superior accelerating effect of water to its ability to form a three-dimensional hydrogen-bond network between the moving parts of the molecular machine. These results may indicate a more general phenomenon that helps explain the function of water as the 'lubricant of life'.</p

Unraveling VEALYL Amyloid Formation Using Advanced Vibrational Spectroscopy and Microscopy

Intermediate species are hypothesized to play an important role in the toxicity of amyloid formation, a process associated with many diseases. This process can be monitored with conventional and two-dimensional infrared spectroscopy, vibrational circular dichroism, and optical and electron microscopy. Here, we present how combining these techniques provides insight into the aggregation of the hexapeptide VEALYL (Val-Glu-Ala-Leu-Tyr-Leu), the B-chain residue 12-17 segment of insulin that forms amyloid fibrils (intermolecularly hydrogen-bonded β-sheets) when the pH is lowered below 4. Under such circumstances, the aggregation commences after approximately an hour and continues to develop over a period of weeks. Singular value decompositions of one-dimensional and two-dimensional infrared spectroscopy spectra indicate that intermediate species are formed during the aggregation process. Multivariate curve resolution analyses of the one and two-dimensional infrared spectroscopy data show that the intermediates are more fibrillar and deprotonated than the monomers, whereas they are less ordered than the final fibrillar structure that is slowly formed from the intermediates. A comparison between the vibrational circular dichroism spectra and the scanning transmission electron microscopy and optical microscope images shows that the formation of mature fibrils of VEALYL correlates with the appearance of spherulites that are on the order of several micrometers, which give rise to a "giant" vibrational circular dichroism effect

Two-Dimensional Vibrational Spectroscopy of Rotaxane-Based Molecular Machines

It has recently become possible to synthesize mechanical devices the size of a single molecule. Although it is tempting to regard such molecular machines as nanoscale versions of their macroscopic analogs, many notions from macroscopic mechanics no longer apply at a molecular level. For instance, the concept of viscous friction is meaningless for a molecular machine because the size of the solvent molecules that cause the friction is comparable to that of the machine itself. Furthermore, in many cases, the interactions between a molecular machine and its surroundings are comparable to the force driving the machine. As a result, a certain amount of intrinsic randomness exists in the motion of molecular machines, and the details of their mechanics are largely unknown. For a detailed understanding of the mechanical behavior of molecular machines, experiments that probe their motion on an ultrafast time scale, such as two-dimensional (2D) vibrational spectroscopy, are essential. This method uses coupling between vibrational modes in a molecule to investigate the molecular conformation. The coupling shows up as off-diagonal peaks in a 2D graph of the vibrational response of the molecule, analogous to the spin coupling observed in multidimensional NMR spectroscopy. Both spin coupling and vibrational coupling are sensitive probes of the molecular conformation, but 2D vibrational spectroscopy shows orders of magnitude better time resolution than NMR. In this Account, we use 2D vibrational spectroscopy to study molecular machines based on rotaxanes. These devices consist of a linear thread and a macrocycle that is noncovalently locked onto the thread. In the rotaxanes we study, the macrocycle and the thread both contain CO and NH groups. By determining the coupling between the stretching modes of these goups from the cross peaks in the 2D spectrum, we directly and quantitatively probe the relative position and orientation of the macrocycle and the thread for both a small model rotaxane and a rotaxane-based molecular shuttle. Our results demonstrate the feasibility of using time-resolved 2D-IR experiments to measure externally triggered structural changes of molecular devices with subpicosecond time resolution. We can observe each of the elementary events that underlie the mechanical motion separately. With this ability to investigate the nature of the mechanical motions at the molecular level and with unprecedented time resolution, we expect that 2D-IR spectroscopy on molecular machines will lead to new insights into their function

Amplifying vibrational circular dichroism by manipulation of the electronic manifold

Vibrational circular dichroism is a powerful technique to study the stereochemistry of chiral molecules, but often suffers from small signal intensities. Electrochemical modulation of the energies of the electronically excited state manifold is now demonstrated to lead to an order of magnitude enhancement of the differential absorption. Quantum-chemical calculations show that increased mixing between ground and excited states is at the origin of this amplification

Operation mechanism of a molecular machine revealed using time-resolved vibrational spectroscopy

Rotaxanes comprise macrocycles that can shuttle between docking stations along an axle. We explored the nanosecond shuttling mechanism by reversing the relative binding affinities of two stations through ultraviolet-induced transient reduction. We monitored the ensuing changes in the CO-stretching bands of the two stations and the shuttling macrocycle by means of an infrared probing pulse. Because hydrogen-bond scission and formation at the initial and final stations led to well-resolved changes in the respective CO-stretch frequencies, the departure and arrival of the macrocycle could be observed separately. We found that the shuttling involves two steps: thermally driven escape from the initial station, followed by rapid motion along the track ending either at the initial or final station. By varying the track’s length, we found that the rapid motion approximates a biased one-dimensional random walk. However, surprisingly, the direction of the overall motion is opposite that of the bias

Transient two-dimensional vibrational spectroscopy of an operating molecular machine

Synthetic molecular machines are promising building blocks for future nanoscopic devices. However, the details of their mechanical behaviour are in many cases still largely unknown. A deeper understanding of mechanics at the molecular level is essential for the design and construction of complex nanodevices. Here, we show that transient two-dimensional infrared (T2DIR) spectroscopy makes it possible to monitor the conformational changes of a translational molecular machine during its operation. Translation of a macrocyclic ring from one station to another on a molecular thread is initiated by a UV pulse. The arrival of the shuttling macrocycle at the final station is visible from a newly appearing cross peak between these two moieties. To eliminate spectral congestion in the T2DIR spectra, we use a subtraction method applicable to many other complex molecular systems. The T2DIR spectra indicate that the macrocycle adopts a boat-like conformation at the final station, which contrasts with the chair-like conformation at the initial station