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The marine cyanobacteriumProchlorococcusis a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes typically encode two different types of FutA iron binding proteins: periplasmic FutA2 ABC transporter subunits bind ferric (Fe3+), while cytosolic FutA1 binds ferrous (Fe2+). Owing to their small size and their economized genomeProchlorococcusecotypes typically possess a singlefutAgene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a solvent molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell was consistent with an overall charge-neutral ferric binding state in structures determined by neutron diffraction and serial femtosecond crystallography.Conventional rotation X-ray crystallography using a home source revealed X-ray induced photoreduction of the iron center with observation of the ferrous binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral ferrous binding site. Room temperature dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between ferric and ferrous states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of theProchlorococcusFutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.Significance StatementOceanic primary production by marine cyanobacteria is a main contributor to carbon and nitrogen fixation.Prochlorococcusis the most abundant photosynthetic organism on Earth, with an annual carbon fixation comparable to the net global primary production from agriculture. Its remarkable ecological success is based on the ability to thrive in low nutrient waters. To manage iron limitation,Prochlorococcuspossesses the FutA protein for iron uptake and homeostasis. We reveal a switch in the FutA protein that allows it to accommodate binding of iron in either the ferric (Fe3+) or ferrous (Fe2+) state using structural biology techniques at room temperature and provide a plausible mechanism for FutA as a bifunctional redox state sensing protein.

Neutron crystallography has been used to elucidate the protonation states for the enhanced green fluorescent protein, which has revolutionized imaging technologies. The structure has a deprotonated hydroxyl group in the fluorescent chromophore. Also, the protonation states of His148 and Thr203, as well as the orientation of a critical water molecule in direct contact with the chromophore, could be determined. The results demonstrate that the deprotonated hydroxyl group in the chromophore and the nitrogen atom ND1 in His148 are charged negatively and positively, respectively, forming an ion pair. The position of the two deuterium atoms in the critical water molecule appears to be displaced slightly toward the acceptor oxygen atoms according to their omit maps. This displacement implies the formation of an intriguing electrostatic potential realized inside of the protein. Our findings provide new insights into future protein design strategies along with developments in quantum chemical calculations.

Applied Sciences 11(11), 5135 (2021). doi:10.3390/app11115135 special issue: "Special Issue "Recent Advances in Small-Angle Neutron Scattering" / Special Issue Editor Dr. Sebastian Jaksch", Published by MDPI, Basel

The isothermal crystallization kinetics of hen-egg-white Lysozyme has been investigated by means of a time-resolved neutron diffraction experiment for almost 3 days, starting from a supersaturated solution of Lysozyme (30 mg/ml, 3 wt% NaCl, pD 4.75 at 298 K) until the growth of crystals, in order to have complementary information about the crystallization kinetics (Heigl et al., 2018). The temporal evolution of the intensity of the Bragg peaks, observed in the neutron diffraction images when a single crystal appears, has been studied. Simultaneously, the analysis of the small angle neutron scattering curves from the Lysozyme solution, during the crystallization process, has been performed. A correlated behaviour between the decrease of the Lysozyme concentration and the increase of the crystallization fraction was observed. The crystallization kinetics was described by means of the Johnson-Mehl-Avrami-Kolmogorov model and parameters compatible with our previous Lysozyme crystallization study have been found. The Lysozyme crystallization under constant neutron flux underlines the known strength of neutrons in studying biological samples without causing radiation damage.

β-casein undergoes a reversible endothermic self-association, forming protein micelles of limited size. In its functional state, a single β-casein monomer is unfolded, which creates a high structural flexibility, supposed to play a major role in preventing the precipitation of calcium phosphate particles. We characterize the structural flexibility in terms of nano-second molecular motions, depending on the temperature by quasi-elastic neutron scattering. Our major questions are: Does the self- association reduce the chain flexibility? How does the dynamic spectrum of disordered caseins differ from a compactly globular protein? How does the dynamic spectrum of β-casein in solution differ from that of a protein in hydrated powder states? We report on two relaxation processes on a nano-second and a sub-nano-second time scale for β-casein in solution. Both processes are analyzed by Brownian Oscillator model, by which the spring constant can be defined in the isotropic parabolic potential. The slower process, which is analyzed by neutron spin echo, seems a characteristic feature of the unfolded structure. It requires bulk solvent and is not seen in hydrated protein powders. The faster process, which is analyzed by neutron backscattering, has a smaller amplitude and requires hydration water, which is also observed with folded proteins in the hydrated state. The self-association had no significant influence on internal relaxation, and thus a β-casein protein monomer flexibility is preserved in the micelle. We derive spring constants of the faster and slower motions of β-caseins in solution, and compared them with those of some proteins in various states; folded or hydrated powder.

Bromodomain-containing protein 4 (BRD4) recognizes the acetylated lysine of histone H4 via its bromodomains, leading to the recruitment of positive transcription elongation factor b. Small molecules that inhibit BRD4 have potential as anticancer agents by leading to the downregulation of specific oncogenes. Using X-ray crystallographic screening, we identified the BRD4 inhibitory activity of isoliquiritigenin (ISL), a natural chalcone found in licorice. Structural analysis revealed that ISL bound to BRD4 with a novel binding mode and squeezed out one of the six conserved water molecules that form a strong hydrogen bond network. The thermodynamic analysis revealed that the binding of ISL is enthalpy driven, suggesting that strong hydrogen bonds would compensate for the desolvation penalty. Neutron protein crystallography further suggested that the favorable binding enthalpy originates from the stabilization and optimization of the hydrogen bond network of the conserved water molecules. Here, we describe the novelty and potential of ISL as a template for new BRD4 inhibitors.

Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IX mimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IX mimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.

In redox metalloenzymes, the process of electron transfer often involves the concerted movement of a proton. These processes are referred to as proton-coupled electron transfer, and they underpin a wide variety of biological processes, including respiration, energy conversion, photosynthesis, and metalloenzyme catalysis. The mechanisms of proton delivery are incompletely understood, in part due to an absence of information on exact proton locations and hydrogen bonding structures in a bona fide metalloenzyme proton pathway. Here, we present a 2.1-Å neutron crystal structure of the complex formed between a redox metalloenzyme (ascorbate peroxidase) and its reducing substrate (ascorbate). In the neutron structure of the complex, the protonation states of the electron/proton donor (ascorbate) and all of the residues involved in the electron/proton transfer pathway are directly observed. This information sheds light on possible proton movements during heme-catalyzed oxygen activation, as well as on ascorbate oxidation.

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Å resolution) and X-ray (1.1 Å resolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%-75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.

Hydrogen bonds are key interactions determining protein-ligand binding affinity and therefore fundamental to any biological process. Unfortunately, explicit structural information about hydrogen positions and thus H-bonds in protein-ligand complexes is extremely rare and similarly the important role of water during binding remains poorly understood. Here, we report on neutron structures of trypsin determined at very high resolutions ≤1.5 Å in uncomplexed and inhibited state complemented by X-ray and thermodynamic data and computer simulations. Our structures show the precise geometry of H-bonds between protein and the inhibitors N-amidinopiperidine and benzamidine along with the dynamics of the residual solvation pattern. Prior to binding, the ligand-free binding pocket is occupied by water molecules characterized by a paucity of H-bonds and high mobility resulting in an imperfect hydration of the critical residue Asp189. This phenomenon likely constitutes a key factor fueling ligand binding via water displacement and helps improving our current view on water influencing protein–ligand recognition., Trypsin is a serine protease. Here the authors present the high resolution X-ray and neutron diffraction structures of uncomplexed and inhibitor bound trypsin that provide insights into the geometry of H-bonds in the active site of the enzyme and molecular dynamics simulations reveal the kinetics of ligand binding induced desolvation.

Using lysozyme as a crystallization model, existing intermediate clusters and aggregates have been previously identified as fractal systems using light scattering techniques. However, this has not been confirmed with neutron or X-ray scattering directly. In this work, we attempt to deepen our knowledge of the role of the fractal clusters during the crystallization process by following the evolution of the fractal dimension df from the early stage of the nucleation process. Indeed, three different scattering techniques have been used simultaneously on the same sample: dynamic light scattering, small-angle neutron scattering, and static light scattering. We focused on the optimal batch crystallization condition in order to obtain large crystals (30 mg/mL lysozyme concentration and 3 wt % sodium chloride at pD 4.75 at 298 K). The selected temperature reduces the nucleation speed allowing us to investigate in detail the very early stage of the crystallization process. A direct temporal change of the fractal d...

The medically important drug target galectin-3 binds galactose-containing moieties on glycoproteins through an intricate pattern of hydrogen bonds to a largely polar surface-exposed binding site. All successful inhibitors of galectin-3 to date have been based on mono- or disaccharide cores closely resembling natural ligands. A detailed understanding of the H-bonding networks in these natural ligands will provide an improved foundation for the design of novel inhibitors. Neutron crystallography is an ideal technique to reveal the geometry of hydrogen bonds because the positions of hydrogen atoms are directly detected rather than being inferred from the positions of heavier atoms as in X-ray crystallography. We present three neutron crystal structures of the C-terminal carbohydrate recognition domain of galectin-3: the ligand-free form and the complexes with the natural substrate lactose and with glycerol, which mimics important interactions made by lactose. The neutron crystal structures reveal unambiguously the exquisite fine-tuning of the hydrogen bonding pattern in the binding site to the natural disaccharide ligand. The ligand-free structure shows that most of these hydrogen bonds are preserved even when the polar groups of the ligand are replaced by water molecules. The protonation states of all histidine residues in the protein are also revealed and correlate well with NMR observations. The structures give a solid starting point for molecular dynamics simulations and computational estimates of ligand binding affinity that will inform future drug design.

By combining the normal practice for X-ray crystallography of collecting diffraction data at 100K with neutron crystallography the structures of cryo-trapped enzyme intermediates have been determined, revealing the positions of the previously hidden hydrogens that are essential to a better understanding of the involved mechanism.

By combining the normal practice for X-ray crystallography of collecting diffraction data at 100 K with neutron crystallography the structures of cryo-trapped enzyme intermediates have been determined, revealing the positions of the previously hidden hydrogens that are essential to a better understanding of the involved mechanism.

Heterotrimeric glutamine amidotransferase CAB (GatCAB) possesses an ammonia-self-sufficient mechanism in which ammonia is produced and used in the inner complex by GatA and GatB, respectively. The X-ray structure of GatCAB revealed that the two identified active sites of GatA and GatB are markedly distant, but are connected in the complex by a channel of 30 Å in length. In order to clarify whether ammonia is transferred through this channel in GatCAB by visualizing ammonia, neutron diffraction studies are indispensable. Here, GatCAB crystals were grown to approximate dimensions of 2.8 × 0.8 × 0.8 mm (a volume of 1.8 mm3) with the aid of a polymer using microseeding and macroseeding processes. Monochromatic neutron diffraction data were collected using the neutron single-crystal diffractometer BIODIFF at the Heinz Maier-Leibnitz Zentrum, Germany. The GatCAB crystals belonged to space group P212121, with unit-cell parameters a = 74.6, b = 94.5, c = 182.5 Å and with one GatCAB complex (molecular mass 119 kDa) in the asymmetric unit. This study represented a challenge in current neutron diffraction technology.

Lactobacillus brevis alcohol dehydrogenase (LbADH) is a well studied homotetrameric enzyme which catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. LbADH is stable and enzymatically active at elevated temperatures and accepts a broad range of substrates, making it a valuable tool in industrial biocatalysis. Here, the expression, purification and crystallization of LbADH to generate large, single crystals with a volume of up to 1 mm3 suitable for neutron diffraction studies are described. Neutron diffraction data were collected from an H/D-exchanged LbADH crystal using the BIODIFF instrument at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany to a resolution d min of 2.15 Å in 16 days. This allowed the first neutron crystal structure of LbADH to be determined. The neutron structure revealed new details of the hydrogen-bonding network originating from the ion-binding site of LbADH and provided new insights into the reasons why divalent magnesium (Mg2+) or manganese (Mn2+) ions are necessary for its activity. X-ray diffraction data were obtained from the same crystal at the European Synchrotron Radiation Facility (ESRF), Grenoble, France to a resolution d min of 1.48 Å. The high-resolution X-ray structure suggested partial occupancy of Mn2+ and Mg2+ at the ion-binding site. This is supported by the different binding affinity of Mn2+ and Mg2+ to the tetrameric structure calculated via free-energy molecular-dynamics simulations.

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.

Farnesyl pyrophosphate synthase (FPPS) catalyzes the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate to FPP and is known to be a molecular target of osteoporosis drugs, such as risedronate (RIS), which is a nitrogen-containing bisphosphonate. The protonation states and hydration structure of RIS bound to FPPS were determined by neutron protein crystallography, which allows direct visualization of hydrogens and deuteriums. The structure analysis revealed that the phosphate groups of RIS were fully deprotonated with the abnormally decreased pKa, and that the roles of E93 and D264 consisted of canceling the extra negative charges upon the binding of ligands. Collectively, our neutron structures provided insights into the physicochemical properties during the bisphosphonate binding event.

Carbohydrate-binding modules (CBMs) are discrete parts of carbohydrate-hydrolyzing enzymes that bind specific types of carbohydrates. Ultra high-resolution X-ray crystallographic studies of CBMs have helped to decipher the basis for specificity in carbohydrate–protein interactions. However, additional studies are needed to better understand which structural determinants confer which carbohydrate-binding properties. To address these issues, neutron crystallographic studies were initiated on one experimentally engineered CBM derived from a xylanase, X-2 L110F, a protein that is able to bind several different plant carbohydrates such as xylan, β-glucan and xyloglucan. This protein evolved from a CBM present in xylanase Xyn10A ofRhodothermus marinus. The protein was complexed with a branched xyloglucan heptasaccharide. Large single crystals of hydrogenous protein (∼1.6 mm3) were grown at room temperature and subjected to H/D exchange. Both neutron and X-ray diffraction data sets were collected to 1.6 Å resolution. Joint neutron and X-ray refinement usingphenix.refineshowed significant density for residues involved in carbohydrate binding and revealed the details of a hydrogen-bonded water network around the binding site. This is the first report of a neutron structure of a CBM and will add to the understanding of protein–carbohydrate binding interactions.

The use of cryocooling in neutron diffraction has been hampered by several technical challenges, such as the need for specialized equipment and techniques. This article reports the recent development and deployment of equipment and strategies that allow routine neutron data collection on cryocooled crystals using off-the-shelf components. This system has several advantages compared to a closed displex cooling system, such as fast cooling coupled with easier crystal mounting and centering. The ability to routinely collect cryogenic neutron data for analysis will significantly broaden the range of scientific questions that can be examined by neutron protein crystallography. Cryogenic neutron data collection for macromolecules has recently become available at the new Biological Diffractometer BIODIFF at the FRM II and the Macromolecular Diffractometer (MaNDi) at the Spallation Neutron Source, Oak Ridge National Laboratory. To evaluate the benefits of a cryocooled neutron structure, a full neutron data set was collected on the BIODIFF instrument on a Toho-1 β-lactamase structure at 100 K.

The enzyme 2-keto-3-deoxy-9-O-phosphonononic acid phosphatase (KDN9P phosphatase) functions in the pathway for the production of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, a sialic acid that is important for the survival of commensal bacteria in the human intestine. The enzyme is a member of the haloalkanoate dehalogenase superfamily and represents a good model for the active-site protonation state of family members. Crystals of approximate dimensions 1.5 × 1.0 × 1.0 mm were obtained in space group P2(1)2(1)2, with unit-cell parameters a = 83.1, b = 108.9, c = 75.7 Å. A complete neutron data set was collected from a medium-sized H/D-exchanged crystal at BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany in 18 d. Initial refinement to 2.3 Å resolution using only neutron data showed significant density for catalytically important residues.

The mechanism by which class A β-lactamases hydrolyze β-lactam antibiotics has been the subject of intensive investigation using many different experimental techniques. Here, we report on the novel use of both neutron and high resolution x-ray diffraction to help elucidate the identity of the catalytic base in the acylation part of the catalytic cycle, wherein the β-lactam ring is opened and an acyl-enzyme intermediate forms. To generate protein crystals optimized for neutron diffraction, we produced a perdeuterated form of the Toho-1 β-lactamase R274N/R276N mutant. Protein perdeuteration, which involves replacing all of the hydrogen atoms in a protein with deuterium, gives a much stronger signal in neutron diffraction and enables the positions of individual deuterium atoms to be located. We also synthesized a perdeuterated acylation transition state analog, benzothiophene-2-boronic acid, which was also isotopically enriched with 11B, as 10B is a known neutron absorber. Using the neutron diffraction data from the perdeuterated enzyme-inhibitor complex, we were able to determine the positions of deuterium atoms in the active site directly rather than by inference. The neutron diffraction results, along with supporting bond-length analysis from high resolution x-ray diffraction, strongly suggest that Glu-166 acts as the general base during the acylation reaction. Background: Antibiotic resistance from extended-spectrum β-lactamases (ESBLs) makes infections more dangerous and difficult to treat. Results: Neutron and x-ray crystal structures were determined for an ESBL in complex with an acylation transition state analog. Conclusion: Glu-166 is implicated as the general base in the acylation reaction. Significance: Understanding the catalytic mechanism of β-lactamases will lead to improved antibiotics and β-lactamase inhibitors.

The stratum corneum (SC) provides the main barrier properties in native skin. The barrier function is attributed to the intercellular lipids, forming continuous multilamellar membranes. In this study, SC lipid membranes in model ratios were enriched with deuterated lipids in order to investigate structural and dynamical properties by neutron diffraction and 2H solid-state NMR spectroscopy. Further, the effect of the penetration enhancer isopropyl myristate (IPM) on the structure of a well-known SC lipid model membrane containing synthetically derived methyl-branched ceramide [EOS], ceramide [AP], behenic acid and cholesterol (23/10/33/33 wt%) was investigated. IPM supported the formation of a single short-periodicity phase (SPP), in which we determined the molecular organization of CER[AP] and CER[EOS]-br for the first time. Furthermore, the thermotropic phase behavior of the lipid system was analyzed by additional neutron diffraction studies as well as by 2H solid-state NMR spectroscopy, covering temperatures of 32 °C (physiological skin temperature), 50 °C, and 70 °C with a subsequent cooldown back to skin temperature. Both techniques revealed a phase transition and a hysteresis effect. During the cooldown, Bragg peaks corresponding to a long-periodicity phase (LPP) appeared. Additionally, 2H NMR revealed that the IPM molecules are isotopic mobile at all temperatures.

HSP70 belongs to the heat-shock protein family and binds to unfolded proteins, driven by ATP hydrolysis, in order to prevent aggregation. Previous X-ray crystallographic analyses of HSP70 have shown that HSP70 binds to ADP with internal water molecules. In order to elucidate the role of the water molecules, including their H/D atoms, a neutron diffraction study of the human HSP70 ATPase domain was initiated. Deuterated large crystals of the HSP–ADP complex (1.2–1.8 mm3) were successfully grown by large-scale crystallization, and a neutron diffraction experiment at BIODIFF resulted in diffraction to a maximum resolution of 2.2 Å. After data reduction, the overall completeness, R meas and average I/σ(I) were 90.4%, 11.7% and 8.1, respectively, indicating that the data set was sufficient to visualize H and D atoms.

MTAN (5′-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple proton-transfer events. Of particular interest is the protonation state of residue D198, which possesses a pKa above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wild-type HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deaza-adenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.

The stratum corneum (SC) provides the main barrier properties in native skin. The barrier function is attributed to the intercellular lipids, forming continuous multilamellar membranes. In this study, SC lipid membranes in model ratios were enriched with deuterated lipids in order to investigate structural and dynamical properties by neutron diffraction and

Nitrogen-containing bisphosphonates (N-BPs), such as risedronate and zoledronate, are currently used as a clinical drug for bone-resorption diseases and are potent inhibitors of farnesyl pyrophosphate synthase (FPPS). X-ray crystallographic analyses of FPPS with N-BPs have revealed that N-BPs bind to FPPS with three magnesium ions and several water molecules. To understand the structural characteristics of N-BPs bound to FPPS, including H atoms and hydration by water, neutron diffraction studies were initiated using BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ). FPPS–risedronate complex crystals of approximate dimensions 2.8 × 2.5 × 1.5 mm (∼3.5 mm3) were obtained by repeated macro-seeding. Monochromatic neutron diffraction data were collected to 2.4 Å resolution with 98.4% overall completeness. Here, the first successful neutron data collection from FPPS in complex with N-BPs is reported.

The neutron single crystal diffractometer BIODIFF at the research reactor Heinz Maier-Leibnitz (FRM II) is especially designed to collect data from crystals with large unit cells. The main field of application is the structural analysis of proteins, especially the determination of hydrogen atom positions. BIODIFF is a joint project of the Jülich Centre for Neutron Science (JCNS) and the FRM II. BIODIFF is designed as a monochromatic instrument with a narrow wavelength spread of less than 3 %. To cover a large solid angle the main detector of BIODIFF consists of a neutron imaging plate in a cylindrical geometry with online read-out capability. BIODFF is equipped with a standard Oxford Cryosystem “Cryostream 700+” which allows measurements at 100 K. A new kappa goniometer head was added recently. This allows an automated tilting of the crystal in order to increase the completeness of the data set when recording another set of frames in the tilted geometry. Typical scientific questions addressed are the determination of protonation states of amino acid side chains in proteins and the characterization of the hydrogen bonding networks between the protein active centre and an inhibitor or substrate. Picking out some recent highlights from measurements at BIODIFF it will be shown how the method of neutron protein crystallography could be used to answer mechanistic questions in enzymatic processes or help to improve inhibitor fragment screening.

Intramolecular charge transfer in aminobenzonitrile (DIABN) single crystals is studied by a combination of ultrafast techniques. The intramolecular dynamics is probed by time-resolved spectroscopy in the UV/Vis and mid-IR. The intermolecular structural response is investigated by transient X-ray scattering and transmission experiments. A theoretical analysis shows that diffuse scattering and dipole solvation determine the X-ray data. The observed dynamics results from charge transfer reaction leading to strong local changes of molecular dipole moments and related structural rearrangements in the crystal.

The photodynamics of single crystals of 4-(diisopropylamino)benzonitrile (DIABN) have been studied by femtosecond UV/Vis and mid-IR absorption spectroscopy. The observed spectroscopic response, especially in the UV/Vis, strongly depends on the optical excitation conditions. For the excitation light in resonance with the absorption of DIABN, broad and structureless transient spectra are observed, which decay non-exponentially within pico- to nanoseconds. For off-resonance excitation in the very red wing of the absorption band a stimulated emission signal is detected, which decays in ~10 ps matching the known behavior of the non-interacting chromophore in solution. The 10 ps decay is due to an intramolecular charge transfer process which is supported by femtosecond IR spectroscopy. The observed dependence on the excitation wavelength is assigned to a change in the density of excited chromophores and therefore bears relevance for time resolved X-ray diffraction experiments.

Ultrafast IR-pump IR-probe spectroscopy is used to study the relaxation dynamics of propionic acid dimers. The spectral changes in the regions of CO-stretching at ∼1700 cm −1 , OH and CH-bending modes around 1400 cm −1 are investigated after excitation in the range of the CH- and CO-stretching modes. A strong coupling of the OH-stretch vibration to CO-stretching and OH/CH-bending modes is found. The intramolecular redistribution of vibrational energy takes place on a 1 ps time-scale. A further cooling process and energy transfer to the surrounding occur with ∼12 ps. The dynamics depend on the wavelength of the IR-excitation and point to a transient breaking of one of the two H-bonds of the propionic acid dimers.

The prototype operation of an ultrafast write-readout-erase-readout cycle of an all-optical system based on photochromic indolylfulgides is demonstrated. In the employed dye the molecular structure is switched using light of different wavelengths between two thermally stable states to allow binary encoding of information. Non-destructive readout of the bit states using infrared light completes the scheme of an all-optical memory. For ultrafast operation femtosecond light pulses are applied and it is demonstrated that two consecutive write/erase processes separated by less than 4 ps still allow the defined readout of the bit state. The short time between the write/erase and readout actions demonstrates that an all-optical data storage system based on indolylfulgides may be operated at memory clock rates that exceed 250 GHz.

Femtosecond time-resolved infrared spectroscopy was used to study the formation of cyclobutane dimers in the all-thymine oligodeoxynucleotide (dT)18by ultraviolet light at 272 nanometers. The appearance of marker bands in the time-resolved spectra indicates that the dimers are fully formed ∼1 picosecond after ultraviolet excitation. The ultrafast appearance of this mutagenic photolesion points to an excited-state reaction that is approximately barrierless for bases that are properly oriented at the instant of light absorption. The low quantum yield of this photoreaction is proposed to result from infrequent conformational states in the unexcited polymer, revealing a strong link between conformation before light absorption and photodamage.

A light-switchable peptide is transformed with ultrashort pulses from a β-hairpin to an unfolded hydrophobic cluster and vice versa. The structural changes are monitored by mid-IR probing. Instantaneous normal mode analysis with a Hamiltonian combining density functional theory with molecular mechanics is used to interpret the absorption transients. Illumination of the β-hairpin state triggers an unfolding reaction that visits several intermediates and reaches the unfolded state within a few nanoseconds. In this unfolding reaction to the equilibrium hydrophobic cluster conformation, the system does not meet significant barriers on the free-energy surface. The reverse folding process takes much longer because it occurs on the time scale of 30 μs. The folded state has a defined structure, and its formation requires an extended search for the correct hydrogen-bond pattern of the β-strand.

The photo-induced trans–cis isomerization of the azobenzene derivative 4-nitro-4 0 -(dimethylamino)azobenzene in polar solution was studied by femtosecond UV/Vis and IR spectroscopy. The UV/Vis experiment reveals two excited state processes; the slower one (1 ps) is the internal conversion to the ground state. The ensuing spectral changes point to vibrational cooling of the nascent cis product and the recovered trans isomer. Judging from the UV/Vis experiment this � 5 ps process seems to terminate the isomerization. The internal conversion and the cooling also find their manifestation in the IR experiment. In addition slower spectral changes lasting until � 50 ps are detected. These slow IR responses are compared with the behavior of a non-isomerizing analogue, para-N,N-dimethyl-nitroaniline. An origin for this discrepancy is suggested and potential molecular processes causing the slow IR response are discussed. 2007 Elsevier B.V. All rights reserved.

The catalytic mechanism of class A β-lactamases is often debated due in part to the large number of amino acids that interact with bound β-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type β-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 β-lactamase with the antibiotic cefotaxime. The E166A mutant lacks a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzyme's native machinery.

The single crystal diffractometer BIODIFF, which is jointly operated by the Technische Universität München and JCNS, Forschungszentrum Jülich, is designed to handle crystals with large unit cells and is dedicated to the structure determination of biological macromolecules. The main field of application is the neutron structure analysis of proteins, especially the determination of hydrogen atom positions.

The photo-induced ring-opening reaction C → E of a photochromic indolyl-fulgimide is investigated by sub-picosecond pump–probe spectroscopy in the visible and infrared spectral range. The thermally stable C-isomer shows a pronounced absorption in the visible spectral range without spectral overlap with the absorption of the E- and Z-isomer. The comparison of infrared and visible transient absorption data allows to assign the observed exponential 2.1 ps dynamics to the ring-opening reaction and the non-exponential 10–40 ps component to the cooling of the hot molecules. The transient absorption spectra taken 80 ps after photo-excitation are identical with the steady state difference spectra.

Femtosecond IR and Raman techniques are compared with respect to their abilities to monitor vibrational relaxation following ultrafast internal conversion. The study was performed on para -nitroaniline which undergoes sub-picosecond internal conversion to the ground state populating a variety of vibrational modes. The NO 2 stretch vibration at 1315 cm −1 responds to the excitation by a strong red shift, visible in transient IR and Raman experiments. It is due to the off-diagonal anharmonic coupling with other modes. The population of the NO 2 stretch mode itself can only be observed in the (anti-Stokes) Raman spectrum, whereas IR spectroscopy is superior in detecting the coupling induced shift.

Heme enzymes activate oxygen through formation of transient iron-oxo (ferryl) intermediates of the heme iron. A long-standing question has been the nature of the iron-oxygen bond and, in particular, the protonation state. We present neutron structures of the ferric derivative of cytochrome c peroxidase and its ferryl intermediate; these allow direct visualization of protonation states. We demonstrate that the ferryl heme is an Fe(IV)=O species and is not protonated. Comparison of the structures shows that the distal histidine becomes protonated on formation of the ferryl intermediate, which has implications for the understanding of O-O bond cleavage in heme enzymes. The structures highlight the advantages of neutron cryo-crystallography in probing reaction mechanisms and visualizing protonation states in enzyme intermediates.

Conformational changes in proteins and peptides can be initiated by diverse processes. This raises the question how the variation of initiation mechanisms is connected to differences in folding or unfolding processes. In this work structural dynamics of a photoswitchable β-hairpin model peptide were initiated by two different mechanisms: temperature jump (T-jump) and isomerization of a backbone element. In both experiments the structural changes were followed by time-resolved IR spectroscopy in the nanosecond to microsecond range. When the photoisomerization of the azobenzene backbone switch initiated the folding reaction, pronounced absorption changes related to folding into the hairpin structure were found with a time constant of about 16 μs. In the T-jump experiment kinetics with the same time constant were observed. For both initiation processes the reaction dynamics revealed the same strong dependence of the reaction time on temperature. The highly similar transients in the microsecond range show that the peptide dynamics induced by T-jump and isomerization are both determined by the same mechanism and exclude a downhill-folding process. Furthermore, the combination of the two techniques allows a detailed model for folding and unfolding to be presented: The isomerization-induced folding process ends in a transition-state reaction scheme, in which a high energetic barrier of 48 kJ mol(-1) separates unfolded and folded structures.