Evolutionary optimization and long-term stabilization of a white-light seeded two-stage OPCPA seed laser.

Ultrafast laser systems, such as optical parametric chirped pulse amplifiers (OPCPA), are complex tools. Optimizing laser performance for a given application is often plagued by intricate couplings between different output parameters, making simultaneous control of multiple pulse properties difficult. Here, we experimentally demonstrate an autonomous tuning procedure of a white-light seeded two-stage OPCPA using an evolutionary strategy to reliably reach an optimized working point. We use the data collected during the tuning procedure to calibrate a performance model of the laser system, which we then apply to stabilize the intricately coupled laser output energy and spectrum simultaneously. Our approach ensures reliable day-to-day operation at optimized working points without manual tuning. We demonstrate shot-to-shot energy stability of <0.18 % rms, in combination with <25 pm rms wavelength stability and <0.2 % rms bandwidth stability during multi-day operation.

Out-of-plane multilayer-dielectric-grating compressor for ultrafast Ti:sapphire pulses.

Extreme heat loads on optics, in particular the final pulse compression gratings, are a major hurdle to overcome in the ongoing push towards high average power (kW) and high repetition rate (kHz) operation of terawatt-class Ti:sapphire lasers. Multilayer dielectric (MLD) diffraction gratings have been suggested as a potential alternative to traditionally gold-coated compressor gratings, which are plagued by high energy absorption in the top gold layer. However, to support the required bandwidth (and ultimately the desired pulse duration) with MLD gratings, the gratings have to be operated in an out-of-plane geometry near the Littrow angle. Here, we report on the design of an MLD-based out-of-plane test compressor and a matching custom stretcher. We present a full characterization of the MLD compressor, focusing on its spectral transmission and the significance of laser pulse polarization in the out-of-plane geometry. To demonstrate compression of 40 µJ pulses centered at 800 nm wavelength to 26 fs pulse duration, we use the compressor with an MLD and gold grating configuration, and fully characterize the compressed pulses. Extrapolating our results indicates that MLD-grating-based out-of-plane compressors can support near-transform-limited pulses with sub-30 fs duration and good quality, demonstrating the viability of this concept for kW-level ultrafast Ti:sapphire laser systems.

Frequency-doubled Q-switched 4 × 4 multicore fiber laser system.

Frequency doubling of a Q-switched Yb-doped rod-type 4 × 4 multicore fiber (MCF) laser system is reported. A second harmonic generation (SHG) efficiency of up to 52% was achieved with type I non-critically phase-matched lithium triborate (LBO), with a total SHG pulse energy of up to 17 mJ obtained at 1 kHz repetition rate. The dense parallel arrangement of amplifying cores into a shared pump cladding enables a significant increase in the energy capacity of active fibers. The frequency-doubled MCF architecture is compatible with high-repetition-rate and high-average-power operation and may provide an efficient alternative to bulk solid-state systems as pump sources for high-energy titanium-doped sapphire lasers.

Spatio-spectral couplings in saturated collinear OPCPA.

Ultrafast laser pulses featuring both high spatio-temporal beam quality and excellent energy stability are crucial for many applications. Here, we present a seed laser with high beam quality and energy stability, based on a collinear optical parametric chirped pulse amplification (OPCPA) stage, delivering 46 µJ pulses with a 25 fs Fourier limit at 1 kHz repetition rate. While saturation of the OPCPA stage is necessary for achieving the highest possible energy stability, it also leads to a degradation of the beam quality. Using simulations, we show that spectrally dependent, rotationally symmetric aberrations dominate the collinear OPCPA in saturation. We experimentally characterize these aberrations and then remove distinct spatial frequencies to greatly improve the spectral homogeneity of the beam quality, while keeping an excellent energy stability of 0.2 % rms measured over 70 hours.

Optimal Beam Loading in a Laser-Plasma Accelerator.

Applications of laser-plasma accelerators demand low energy spread beams and high-efficiency operation. Achieving both requires flattening the accelerating fields by controlled beam loading of the plasma wave. Here, we optimize the generation of an electron bunch via localized ionization injection, such that the combination of injected current profile and averaged acceleration dynamics results in optimal beam loading conditions. This enables the reproducible production of 1.2% rms energy spread bunches with 282 MeV and 44 pC at an estimated energy-transfer efficiency of ∼19%. We correlate shot-to-shot variations to reveal the phase space dynamics and train a neural network that predicts the beam quality as a function of the drive laser.

Description of spatio-temporal couplings from heat-induced compressor grating deformation.

High average power high-intensity laser systems can suffer from a heat-induced deformation of the final compressor gratings, which introduces wavefront aberrations and spatio-temporal couplings to the pulse. Here, we use a simple numerical description, that was first introduced by Li et al. (Appl. Phys. Express, 10, 102702, 2017 and Optics Express, 26, 8453, 2018), to calculate the resulting degradation of the peak intensity and the 3-dimensional deformation of the laser pulse as a function of average power, and verify the results using experimental data. For a typical 100 TW-class laser we find that non-negligible pulse distortions can occur at an average power as low as 2.7 Watts. An open source implementation of our numerical description is available for researchers to estimate the effects of spatio-temporal couplings for their specific laser configuration.

Spectral phase control of interfering chirped pulses for high-energy narrowband terahertz generation.

Highly-efficient optical generation of narrowband terahertz radiation enables unexplored technologies and sciences from compact electron acceleration to charge manipulation in solids. State-of-the-art conversion efficiencies are currently achieved using difference-frequency generation driven by temporal beating of chirped pulses but remain, however, far lower than desired or predicted. Here we show that high-order spectral phase fundamentally limits the efficiency of narrowband difference-frequency generation using chirped-pulse beating and resolve this limitation by introducing a novel technique based on tuning the relative spectral phase of the pulses. For optical terahertz generation, we demonstrate a 13-fold enhancement in conversion efficiency for 1%-bandwidth, 0.361 THz pulses, yielding a record energy of 0.6 mJ and exceeding previous optically-generated energies by over an order of magnitude. Our results prove the feasibility of millijoule-scale applications like terahertz-based electron accelerators and light sources and solve the long-standing problem of temporal irregularities in the pulse trains generated by interfering chirped pulses.

Wavefront degradation of a 200 TW laser from heat-induced deformation of in-vacuum compressor gratings.

High-repetition-rate high-power laser systems induce a high average power heat deposition into the gold-coated diffraction gratings. To study the effects of the thermal expansion of in-vacuum Pyrex gratings on the laser properties, we scan the pulse energy and repetition rate of a 200 TW laser system while monitoring the laser wavefront. Through the measured changes in laser divergence and focusability, we define an average power limit below which the in-vacuum compressor can be used with no degradation of the laser focus quality.

Regulation of Microtubule Assembly by Tau and not by Pin1.

The molecular mechanism by which the microtubule-associated protein (MAP) tau regulates the formation of microtubules (MTs) is poorly understood. The activity of tau is controlled via phosphorylation at specific Ser/Thr sites. Of those phosphorylation sites, 17 precede a proline, making them potential recognition sites for the peptidyl-prolyl isomerase Pin1. Pin1 binding and catalysis of phosphorylated tau at the AT180 epitope, which was implicated in Alzheimer's disease, has been reported to be crucial for restoring tau's ability to promote MT polymerization in vitro and in vivo [1]. Surprisingly, we discover that Pin1 does not promote phosphorylated tau-induced MT formation in vitro, refuting the commonly accepted model in which Pin1 binding and catalysis on the A180 epitope restores the function of the Alzheimer's associated phosphorylated tau in tubulin assembly [1, 2]. Using turbidity assays, time-resolved small angle X-ray scattering (SAXS), and time-resolved negative stain electron microscopy (EM), we investigate the mechanism of tau-mediated MT assembly and the role of the Thr231 and Ser235 phosphorylation on this process. We discover novel GTP-tubulin ring-shaped species, which are detectable in the earliest stage of tau-induced polymerization and may play a crucial role in the early nucleation phase of MT assembly. Finally, by NMR and SAXS experiments, we show that the tau molecules must be located on the surface of MTs and tubulin rings during the polymerization reaction. The interaction between tau and tubulin is multipartite, with a high affinity interaction of the four tubulin-binding repeats, and a weaker interaction with the proline-rich sequence and the termini of tau.

Molecular Mechanism of Pin1-Tau Recognition and Catalysis.

Human peptidyl-prolyl isomerase (PPIase) Pin1 plays key roles in developmental processes, cell proliferation, and neuronal function. Extensive phosphorylation of the microtubule binding protein tau has been implicated in neurodegeneration and Alzheimer's disease. For the past 15years, these two players have been the focus of an enormous research effort to unravel the biological relevance of their interplay in health and disease, resulting in a series of proposed molecular mechanism of how Pin1 catalysis of tau results in biological phenotypes. Our results presented here refute these mechanisms of Pin1 action. Using NMR, isothermal calorimetry (ITC), and small angle x-ray scattering (SAXS), we dissect binding and catalysis on multiple phosphorylated tau with particular emphasis toward the Alzheimer's associated AT180 tau epitope containing phosphorylated THR231 and SER235. We find that phosphorylated (p-) SER235-PRO, but not pTHR231-PRO, is exclusively catalyzed by full-length Pin1 and isolated PPIase domain. Importantly, site-specific measurements of Pin1-catalysis of CDK2/CycA-phosphorylated full-length tau reveal a number of sites that are catalyzed simultaneously with different efficiencies. Furthermore, we show that the turnover efficiency at pSER235 by Pin1 is independent of both the WW domain and phosphorylation on THR231. Our mechanistic results on site-specific binding and catalysis together with the lack of an increase of dephosphorylation rates by PP2A counter a series of previously published models for the role of Pin1 catalysis of tau in Alzheimer's disease. Together, our data reemphasize the complicated scenario between binding and catalysis of multiple phosphorylated tau by Pin1 and the need for directly linking biological phenotypes and residue-specific turnover in Pin1 substrates.

Identification and Characterization of Botulinum Neurotoxin A Substrate Binding Pockets and Their Re-Engineering for Human SNAP-23.

Botulinum neurotoxins (BoNTs) are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs (soluble N-ethyl maleimide sensitive factor attachment protein receptors). However, their serotype A (BoNT/A) that cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25 years. The expansion of its use to a variety of further medical conditions associated with hypersecretion components is prevented partly because the involved SNARE isoforms are not cleaved. Therefore, we examined by mutational analyses the reason for the resistance of human SNAP-23, an isoform of SNAP-25. We show that replacement of 10 SNAP-23 residues with their SNAP-25 counterparts effects SNAP-25-like cleavability. Conversely, transfer of each of the replaced SNAP-23 residues to SNAP-25 drastically decreased the cleavability of SNAP-25. By means of the existing SNAP-25-toxin co-crystal structure, molecular dynamics simulations, and corroborative mutagenesis studies, the appropriate binding pockets for these residues in BoNT/A were characterized. Systematic mutagenesis of two major BoNT/A binding pockets was conducted in order to adapt these pockets to corresponding amino acids of human SNAP-23. Human SNAP-23 cleaving mutants were isolated using a newly established yeast-based screening system. This method may be useful for engineering novel BoNT/A pharmaceuticals for the treatment of diseases that rely on SNAP-23-mediated hypersecretion.

Botulinum neurotoxin G binds synaptotagmin-II in a mode similar to that of serotype B: tyrosine 1186 and lysine 1191 cause its lower affinity.

Botulinum neurotoxins (BoNTs) block neurotransmitter release by proteolyzing SNARE proteins in peripheral nerve terminals. Entry into neurons occurs subsequent to interaction with gangliosides and a synaptic vesicle protein. Isoforms I and II of synaptotagmin were shown to act as protein receptors for two of the seven BoNT serotypes, BoNT/B and BoNT/G, and for mosaic-type BoNT/DC. BoNT/B and BoNT/G exhibit a homologous binding site for synaptotagmin whose interacting part adopts helical structure upon binding to BoNT/B. Whereas the BoNT/B-synaptotagmin-II interaction has been elucidated in molecular detail, corresponding information about BoNT/G is lacking. Here we systematically mutated the synaptotagmin binding site in BoNT/G and performed a comparative binding analysis with mutants of the cell binding subunit of BoNT/B. The results suggest that synaptotagmin takes the same overall orientation in BoNT/B and BoNT/G governed by the strictly conserved central parts of the toxins' binding site. The surrounding nonconserved areas differently contribute to receptor binding. Reciprocal mutations Y1186W and L1191Y increased the level of binding of BoNT/G approximately to the level of BoNT/B affinity, suggesting a similar synaptotagmin-bound state. The effects of the mutations were confirmed by studying the activity of correspondingly mutated full-length BoNTs. On the basis of these data, molecular modeling experiments were employed to reveal an atomistic model of BoNT/G-synaptotagmin recognition. These data suggest a reduced length and/or a bend in the C-terminal part of the synaptotagmin helix that forms upon contact with BoNT/G as compared with BoNT/B and are in agreement with the data of the mutational analyses.

Broad substrate specificity of the amide synthase in S. hygroscopicus--new 20-membered macrolactones derived from geldanamycin.

The amide synthase of the geldanamycin producer, Streptomyces hygroscopicus, shows a broader chemoselectivity than the corresponding amide synthase present in Actinosynnema pretiosum, the producer of the highly cytotoxic ansamycin antibiotics, the ansamitocins. This was demonstrated when blocked mutants of both strains incapable of biosynthesizing 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase starter unit of both natural products, were supplemented with 3-amino-5-hydroxymethylbenzoic acid instead. Unlike the ansamitocin producer A. pretiosum, S. hygroscopicus processed this modified starter unit not only to the expected 19-membered macrolactams but also to ring enlarged 20-membered macrolactones. The former mutaproducts revealed the sequence of transformations catalyzed by the post-PKS tailoring enzymes in geldanamycin biosynthesis. The unprecedented formation of the macrolactones together with molecular modeling studies shed light on the mode of action of the amide synthase responsible for macrocyclization. Obviously, the 3-hydroxymethyl substituent shows similar reactivity and accessibility toward C-1 of the seco-acid as the arylamino group, while phenolic hydroxyl groups lack this propensity to act as nucleophiles in the macrocyclization. The promiscuity of the amide synthase of S. hygroscopicus was further demonstrated by successful feeding of four other m-hydroxymethylbenzoic acids, leading to formation of the expected 20-membered macrocycles. Good to moderate antiproliferative activities were encountered for three of the five new geldanamycin derivatives, which matched well with a competition assay for Hsp90α.

A diversity of assembly mechanisms of a generic amyloid fold.

Protein misfolding and amyloid assembly have long been recognized as being responsible for many devastating human diseases. Recent findings indicate that amyloid assemblies may facilitate crucial biological processes from bacteria to mammals. This review focuses on the mechanistic understanding of amyloid formation, including the transformation of initially innocuous proteins into oligomers and fibrils. The result is a competing folding and assembly energy landscape, which contains a number of routes by which the polypeptide chain can convert its primary sequence into functional structures, dysfunctional assemblies, or epigenetic entities that provide both threats and opportunities in the evolution of life.

Understanding the complex mechanisms of ß2-microglobulin amyloid assembly.

Several protein misfolding diseases are associated with the conversion of native proteins into ordered protein aggregates known as amyloid. Studies of amyloid assemblies have indicated that non-native proteins are responsible for initiating aggregation in vitro and in vivo. Despite the importance of these species for understanding amyloid disease, the structural and dynamic features of amyloidogenic intermediates and the molecular details of how they aggregate remain elusive. This review focuses on recent advances in developing a molecular description of the folding and aggregation mechanisms of the human amyloidogenic protein ß(2)-microglobulin under physiologically relevant conditions. In particular, the structural and dynamic properties of the non-native folding intermediate I(T) and its role in the initiation of fibrillation and the development of dialysis-related amyloidosis are discussed.

The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites.

The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons. Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein. Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid. The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively. Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction. Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites. These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins.

Conformational conversion during amyloid formation at atomic resolution.

Numerous studies of amyloid assembly have indicated that partially folded protein species are responsible for initiating aggregation. Despite their importance, the structural and dynamic features of amyloidogenic intermediates and the molecular details of how they cause aggregation remain elusive. Here, we use ΔN6, a truncation variant of the naturally amyloidogenic protein ß(2)-microglobulin (ß(2)m), to determine the solution structure of a nonnative amyloidogenic intermediate at high resolution. The structure of ΔN6 reveals a major repacking of the hydrophobic core to accommodate the nonnative peptidyl-prolyl trans-isomer at Pro32. These structural changes, together with a concomitant pH-dependent enhancement in backbone dynamics on a microsecond-millisecond timescale, give rise to a rare conformer with increased amyloidogenic potential. We further reveal that catalytic amounts of ΔN6 are competent to convert nonamyloidogenic human wild-type ß(2)m (Hß(2)m) into a rare amyloidogenic conformation and provide structural evidence for the mechanism by which this conformational conversion occurs.

A generic mechanism of beta2-microglobulin amyloid assembly at neutral pH involving a specific proline switch.

Although numerous measurements of amyloid assembly of different proteins under distinct conditions in vitro have been performed, the molecular mechanisms underlying the specific self-association of proteins into amyloid fibrils remain obscure. Elucidating the nature of the events that initiate amyloid formation remains a particularly difficult challenge because of the heterogeneity and transient nature of the species involved. Here, we have used site-directed mutagenesis to create five proline to glycine variants in the naturally amyloidogenic protein beta2-microglobulin (beta2m). One of these variants, P5G, allowed us to isolate and characterise an intermediate containing a non-native trans Pro32 backbone conformation, a feature that is known to be required for amyloid elongation at neutral pH. By analysing oligomerisation and amyloid formation using analytical size-exclusion chromatography, multi-angle static light-scattering, analytical ultracentrifugation, circular dichroism and thioflavin T fluorescence we reveal a pathway for beta2m amyloid assembly at pH 7.5 that does not require the addition of metal ions, detergents, co-solvents or other co-factors that have been used to facilitate amyloid formation at physiological pH and temperature. Assembly is shown to involve the transient formation of a non-native monomer containing a trans P32 backbone conformation. This is followed by the formation of dimeric species and higher molecular mass oligomers that accumulate before the development of amyloid fibrils. On the basis of these results, we propose a generic mechanism for beta2m fibrillogenesis at neutral pH that is consistent with the wide range of published studies of this protein. In this mechanism, amyloid formation is initiated by a specific cis to trans proline switch, the rate of which we show to be controlled by the amino acid sequence proximal to P32 and to the applied solution conditions.

Kinetic characterization of the disulfide bond-forming enzyme DsbB.

DsbB is an integral membrane protein responsible for the de novo synthesis of disulfide bonds in Escherichia coli and many other prokaryotes. In the process of transferring electrons from DsbA to a tightly bound ubiquinone cofactor, DsbB undergoes an unusual spectral transition at approximately 510 nm. We have utilized this spectral transition to study the kinetic cycle of DsbB in detail using stopped flow methods. We show that upon mixing of Dsb-B(ox) and DsbA(red), there is a rapid increase in absorbance at 510 nm (giving rise to a purple solution), followed by two slower decay phases. The rate of the initial phase is highly dependent upon DsbA concentration (k(1) approximately 5 x 10(5) M(-1) s(-1)), suggesting this phase reflects the rate of DsbA binding. The rates of the subsequent decay phases are independent of DsbA concentration (k(2) approximately 2 s(-1); k(3) approximately 0.3 s(-1)), indicative of intramolecular reaction steps. Absorbance measurements at 275 nm suggest that k(2) and k(3) are associated with steps of quinone reduction. The rate of DsbA oxidation was found to be the same as the rate of quinone reduction, suggestive of a highly concerted reaction. The concerted nature of the reaction may explain why previous efforts to dissect the reaction mechanism of DsbB by examining individual pairs of cysteines yielded seemingly paradoxical results. Order of mixing experiments showed that the quinone must be pre-bound to DsbB to observe the purple intermediate as well as for efficient quinone reduction. These results are consistent with a kinetic model for DsbB action in which DsbA binding is followed by a rapid disulfide exchange event. This is followed by quinone reduction, which is rate-limiting in the overall reaction cycle.

Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept.

Botulinum neurotoxins (BoNTs) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery within motoneurons. Complex gangliosides initially bind into a pocket that is conserved among the seven BoNTs and tetanus neurotoxin. Productive neurotoxin uptake also requires protein receptors. The interaction site of the protein receptor within the neurotoxin is currently unknown. We report the identification and characterization of the protein receptor binding site of BoNT/B and BoNT/G. Their protein receptors, synaptotagmins I and II, bind to a pocket at the tip of their H(CC) (C-terminal domain of the C-terminal fragment of the heavy chain) that corresponds to the unique second carbohydrate binding site of tetanus neurotoxin, the sialic acid binding site. Substitution of amino acids in this region impaired binding to synaptotagmins and drastically decreased toxicity at mouse phrenic nerve preparations; CD-spectroscopic analyses evidenced that the secondary structure of the mutated neurotoxins was unaltered. Deactivation of the synaptotagmin binding site by single mutations led to virtually inactive BoNT/B and BoNT/G when assayed at phrenic nerve preparations of complex-ganglioside-deficient mice. Analogously, a BoNT B mutant with deactivated ganglioside and synaptotagmin binding sites lacked appreciable activity at wild-type mouse phrenic nerve preparations. Thus, these data exclude relevant contributions of any cell surface molecule other than one ganglioside and one protein receptor to the entry process of BoNTs, which substantiates the double-receptor concept. The molecular characterization of the synaptotagmin binding site provides the basis for designing a novel class of potent binding inhibitors.