B. Leif Hanson

Common processes drive the thermochemical pretreatment of lignocellulosic biomass

Lignocellulosic biomass, a potentially important renewable organic source of energy and chemical feedstock, resists degradation to glucose in industrial hydrolysis processes and thus requires expensive thermochemical pretreatments. Understanding the mechanism of biomass breakdown during these pretreatments will lead to more efficient use of biomass. By combining multiple probes of structure, sensitive to different length scales, with molecular dynamics simulations, we reveal two fundamental processes responsible for the morphological changes in biomass during steam explosion pretreatment: cellulose dehydration and lignin-hemicellulose phase separation. We further show that the basic driving forces are the same in other leading thermochemical pretreatments, such as dilute acid pretreatment and ammonia fiber expansion.

Ionic-Liquid Induced Changes in Cellulose Structure Associated with Enhanced Biomass Hydrolysis

The effects of varying ionic liquid pretreatment parameters on various sources of lignocellulosic biomass have been studied using X-ray powder diffraction, X-ray fiber diffraction, and compositional analysis. Comparative enzymatic hydrolysis and sugar analysis were used to relate the observed changes in cellulose structure to biomass digestibility. In this study, the factor most clearly associated with enhanced biomass hydrolysis is the conversion of cellulose fibers from the cellulose I to the cellulose II crystal phase.

Direct observation of hydrogen atom dynamics and interactions by ultrahigh resolution neutron protein crystallography

The 1.1 Å, ultrahigh resolution neutron structure of hydrogen/deuterium (H/D) exchanged crambin is reported. Two hundred ninety-nine out of 315, or 94.9%, of the hydrogen atom positions in the protein have been experimentally derived and resolved through nuclear density maps. A number of unconventional interactions are clearly defined, including a potential O─H…π interaction between a water molecule and the aromatic ring of residue Y44, as well as a number of potential C─H…O hydrogen bonds. Hydrogen bonding networks that are ambiguous in the 0.85 Å ultrahigh resolution X-ray structure can be resolved by accurate orientation of water molecules. Furthermore, the high resolution of the reported structure has allowed for the anisotropic description of 36 deuterium atoms in the protein. The visibility of hydrogen and deuterium atoms in the nuclear density maps is discussed in relation to the resolution of the neutron data.

A preliminary time-of-flight neutron diffraction study of Streptomyces rubiginosus D-xylose isomerase.

The metalloenzyme D-xylose isomerase forms well ordered crystals that diffract X-rays to ultrahigh resolution (<1 A). However, structural analysis using X-ray diffraction data has as yet been unable to differentiate between several postulated mechanisms that describe the catalytic activity of this enzyme. Neutrons, with their greater scattering sensitivity to H atoms, could help to resolve this by determining the protonation states within the active site of the enzyme. As the first step in the process of investigating the mechanism of action of D-xylose isomerase from Streptomyces rubiginosus using neutron diffraction, data to better than 2.0 A were measured from the unliganded protein at the Los Alamos Neutron Science Center Protein Crystallography Station. Measurement of these neutron diffraction data represents several milestones: this is one of the largest biological molecules (a tetramer, MW approximately 160 000 Da, with unit-cell lengths around 100 A) ever studied at high resolution using neutron diffraction. It is also one of the first proteins to be studied using time-of-flight techniques. The success of the initial diffraction experiments with D-xylose isomerase demonstrate the power of spallation neutrons for protein crystallography and should provide further impetus for neutron diffraction studies of biologically active and significant proteins. Further data will be measured from the enzyme with bound substrates and inhibitors in order to provide the specific information needed to clarify the catalytic mechanism of this enzyme.

Cryogenic (<20 K) helium cooling mitigates radiation damage to protein crystals

In experiments conducted at the Bio-CARS beamline 14-BM-C (APS, Argonne National Laboratory, USA), Streptomyces rubiginosus D-xylose isomerase (EC 5.3.1.5) crystals were used to test the effect of cryogen temperature on radiation damage. Crystals cooled using a helium cryostat at an 8 K set temperature consistently showed less decay in the signal-to-noise ratio, I/sigma(I), and in average intensity, I, compared with those cooled with a nitrogen cryostat set to 100 K. Multiple crystals grown using ammonium sulfate as precipitant were used at each cryostat set temperature and comparisons were made for crystals of similar size and diffraction resolution. Maximum resolution for the crystals was 1.1-1.3 A, with He at <20 K extending the lifetime of the high-resolution data by >25% compared with crystals cooled with N(2) at 100 K.

New techniques in macromolecular cryocrystallography: macromolecular crystal annealing and cryogenic helium

Cryocrystallography is used today for almost all X-ray diffraction data collection at synchrotron beam lines, with rotating-anode generators, and micro X-ray sources. Despite the widespread use of flash-cooling to place macromolecular crystals in the cryogenic state, its use can ruin crystals, trips to the synchrotron, and sometimes even an entire project. Annealing of macromolecular crystals takes little time, requires no specialized equipment, and can save crystallographic projects that might otherwise end in failure. Annealing should be tried whenever initial flash-cooling causes an unacceptable increase in mosaicity, results in ice rings, fails to provide adequate diffraction quality, or causes a crystal to be positioned awkwardly. Overall, annealing improves the quality of data and overall success rate at synchrotron beam lines. Its use should be considered whenever problems arise with a flash-cooled crystal. Helium is a more efficient cryogen than nitrogen and will deliver lower temperatures. Experiments suggest that when crystals are cooled with He rather than N2, crystals maintain order and high-resolution data are less affected by increased radiation load. Individually or in combination, these two techniques can enhance the success of crystallographic data collection, and their use should be considered essential for high-throughput programs.

X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase): perdeuteration of proteins for neutron diffraction.

The signal-to-noise ratio is one of the limiting factors in neutron macromolecular crystallography. Protein perdeuteration, which replaces all H atoms with deuterium, is a method of improving the signal-to-noise ratio of neutron crystallography experiments by reducing the incoherent scattering of the hydrogen isotope. Detailed analyses of perdeuterated and hydrogenated structures are necessary in order to evaluate the utility of perdeuterated crystals for neutron diffraction studies. The room-temperature X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase) is reported at 2.1 A resolution. Comparison with an independently refined hydrogenated room-temperature structure of DFPase revealed no major systematic differences, although the crystals of perdeuterated DFPase did not diffract neutrons. The lack of diffraction is examined with respect to data-collection and crystallographic parameters. The diffraction characteristics of successful neutron structure determinations are presented as a guideline for future neutron diffraction studies of macromolecules. X-ray diffraction to beyond 2.0 A resolution appears to be a strong predictor of successful neutron structures.

Synthesis, capillary crystallization and preliminary joint X-ray and neutron crystallographic study of Z-­DNA without polyamine at low pH

In order to crystallographically study the hydration of the major groove (convex surface) of Z-DNA, the oligonucleotide d(CGCGCG) has been synthesized. Single crystals were grown by vapor diffusion using the hanging-drop and sitting-drop methods for X-ray studies and by batch crystallization and evaporation within silicon tubes for neutron studies. Hexagonal crystals were obtained without the use of duplex-stabilizing polyamines and at an acid pH. X-ray data collected at room temperature (1.5 angstroms resolution; unit-cell parameters a = 17.90, b = 30.59, c = 44.61 angstroms) and at 100 K (1 angstroms resolution; a = 17.99, b = 30.98, c = 44.07 angstroms) and neutron data collected at room temperature (1.6 angstroms resolution; a = 18.00, b = 31.16, c = 44.88 angstroms) indicate that the DNA is in the Z-form packing in space group P2(1)2(1)2(1).

The Well-Tempered Protein Crystal: Annealing Macromolecular Crystals

Publisher Summary This chapter discusses about the annealing macromolecular crystals. Annealing techniques can overcome increased crystal mosaicity after flash cooling. These techniques, involving the heating and cooling of a crystal, can be referred to as annealing or tempering the macromolecular crystal. The term annealing implies a slow or gradual cooling of material after heating. There are several annealing techniques in use today. Macromolecular crystal annealing (MCA) refers to the specific protocol that introduced the concept of annealing and that has been developed originally with crystals of chromatin structural elements (nucleosome core particle). The crystal is incubated in the buffer at either room temperature or the temperature at which the crystal is grown. The single most important precondition for MCA is that the crystal must be stable in the cryoprotectant during the period of incubation before the second flash cooling. The chapter presents the list of macromolecules for which annealing has been reported. Finally, this chapter elucidates the mode of action of annealing by explaining the results from the annealing experiments in terms of the mosaic-block model of the crystals.

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography.

Significance Most enzymatic reactions involve hydrogen or proton transfer among the enzyme, substrate, and water at physiological pH. Thus, enzyme catalysis cannot be fully understood without accurate mapping of hydrogen atom positions in these macromolecular catalysts. Direct information on the location of hydrogen atoms can be obtained using neutron crystallography. We used neutron crystallography and biomolecular simulation to characterize the initial stage of the glycoside hydrolysis reaction catalyzed by a family 11 glycoside hydrolase. We provide evidence that the catalytic glutamate residue alternates between two conformations bearing different basicities, first to obtain a proton from the bulk solvent, and then to deliver it to the glycosidic oxygen to initiate the hydrolysis reaction. 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.