Zoë Fisher

Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. As such, there is enormous industrial interest in using CA as a bio-catalyst for carbon sequestration and biofuel production. However, to ensure cost-effective use of the enzyme under harsh industrial conditions, studies were initiated to produce variants with enhanced thermostability while retaining high solubility and catalytic activity. Kinetic and structural studies were conducted to determine the structural and functional effects of these mutations. X-ray crystallography revealed that a gain in surface hydrogen bonding contributes to stability while retaining proper active site geometry and electrostatics to sustain catalytic efficiency. The kinetic profiles determined under a variety of conditions show that the surface mutations did not negatively impact the carbon dioxide hydration or proton transfer activity of the enzyme. Together these results show that it is possible to enhance the thermal stability of human carbonic anhydrase II by specific replacements of surface hydrophobic residues of the enzyme. In addition, combining these stabilizing mutations with strategic active site changes have resulted in thermostable mutants with desirable kinetic properties.

Neutron Structure of Human Carbonic Anhydrase II: A Hydrogen-Bonded Water Network “Switch” Is Observed between pH 7.8 and 10.0

The neutron structure of wild-type human carbonic anhydrase II at pH 7.8 has been determined to 2.0 Å resolution. Detailed analysis and comparison to the previously determined structure at pH 10.0 show important differences in the protonation of key catalytic residues in the active site as well as a rearrangement of the H-bonded water network. For the first time, a completed H-bonded network stretching from the Zn-bound solvent to the proton shuttling residue, His64, has been directly observed.

Protein structures by spallation neutron crystallography

The capabilities of the Protein Crystallography Station at Los Alamos Neutron Science Center for determining protein structures by spallation neutron crystallography are illustrated, and the methodological and technological advances that are emerging from the Macromolecular Neutron Crystallography consortium are described.

Structural and kinetic analysis of proton shuttle residues in the active site of human carbonic anhydrase III.

We report the X‐ray crystal structures and rate constants for proton transfer in site‐specific mutants of human carbonic anhydrase III (HCA III) that place a histidine residue in the active‐site cavity: K64H, R67H, and K64H‐R67N HCA III. Prior evidence from the exchange of 18O between CO2 and water measured by mass spectrometry shows each mutant to have enhanced proton transfer in catalysis compared with wild‐type HCA III. However, His64 in K64H and K64H‐R67N HCA III have at most a capacity for proton transfer that is only 13% that of His64 in HCA II. This reduced rate in mutants of HCA III is associated with a constrained side‐chain conformation of His64, which is oriented outward, away from the active‐site zinc in the crystal structures. This conformation appears stabilized by a prominent π stacking interaction of the imidazole ring of His64 with the indole ring of Trp5 in mutants of HCA III. This single orientation of His64 in K64H HCA III predominates also in a double mutant K64H‐R67N HCA III, indicating that the positive charge of Arg67 does not influence the observed conformation of His64 in the crystal structure. Hence, the structures and catalytic activity of these mutants of HCA III containing His64 account only in small part for the lower activity of this isozyme compared with HCA II. His67 in R67H HCA III was also shown to be a proton shuttle residue, having a capacity for proton transfer that was approximately four times that of His64 in K64H HCA III. This is most likely due to its proximity and orientation inward towards the zinc‐bound solvent. These results emphasize the significance of side chain orientation and range of available conformational states as characteristics of an efficient proton shuttle in carbonic anhydrase. Proteins 2007.

Direct Determination of Protonation States of Histidine Residues in a 2 Å Neutron Structure of Deoxy-Human Normal Adult Hemoglobin and Implications for the Bohr Effect

We have investigated the protonation states of histidine residues (potential Bohr groups) in the deoxy form (T state) of human hemoglobin by direct determination of hydrogen (deuterium) positions with the neutron protein crystallography technique. The reversible binding of protons is key to the allosteric regulation of human hemoglobin. The protonation states of 35 of the 38 His residues were directly determined from neutron scattering omit maps, with 3 of the remaining residues being disordered. Protonation states of 5 equivalent His residues--alpha His20, alpha His50, alpha His89, beta His143, and beta His146--differ between the symmetry-related globin subunits. The distal His residues, alpha His58 and beta His63, are protonated in the alpha 1 beta 1 heterodimer and are neutral in alpha 2 beta 2. Buried residue alpha His103 is found to be protonated in both subunits. These distal and buried residues have the potential to act as Bohr groups. The observed protonation states of His residues are compared to changes in their pK(a) values during the transition from the T to the R state and the results provide some new insights into our understanding of the molecular mechanism of the Bohr effect.

High-resolution neutron crystallographic studies of the hydration of the coenzyme cob(II)alamin

The hydration of the coenzyme cob(II)alamin has been studied using high-resolution monochromatic neutron crystallographic data collected at room temperature to a resolution of 0.92 Å on the original D19 diffractometer with a prototype 4° × 64° detector at the high-flux reactor neutron source run by the Institute Laue-Langevin. The resulting structure provides hydrogen-bonding parameters for the hydration of biomacromolecules to unprecedented accuracy. These experimental parameters will be used to define more accurate force fields for biomacromolecular structure refinement. The presence of a hydrophobic bowl motif surrounded by flexible side chains with terminal functional groups may be significant for the efficient scavenging of ligands. The feasibility of extending the resolution of this structure to ultrahigh resolution was investigated by collecting time-of-flight neutron crystallographic data during commissioning of the TOPAZ diffractometer with a prototype array of 14 modular 2° × 21° detectors at the Spallation Neutron Source run by Oak Ridge National Laboratory.

Inhibition of d-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography

D-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni(2+) cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg(2+) ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni(2+) ions occupying the catalytic metal site (M2) were found at two locations, while Mg(2+) in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.

Macromolecular neutron crystallography at the Protein Crystallography Station (PCS)

The Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center is a high-performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. Neutron diffraction is a powerful technique for locating H atoms and can therefore provide unique information about how biological macromolecules function and interact with each other and smaller molecules. Users of the PCS have access to neutron beam time, deuteration facilities, the expression of proteins and the synthesis of substrates with stable isotopes and also support for data reduction and structure analysis. The beamline exploits the pulsed nature of spallation neutrons and a large electronic detector in order to collect wavelength-resolved Laue patterns using all available neutrons in the white beam. The PCS user facility is described and highlights from the user program are presented.

Room-temperature ultrahigh-resolution time-of-flight neutron and X-ray diffraction studies of H/D-exchanged crambin

The room-temperature (RT) X-ray structure of H/D-exchanged crambin is reported at 0.85 Å resolution. As one of the very few proteins refined with anisotropic atomic displacement parameters at two temperatures, the dynamics of atoms in the RT and 100 K structures are compared. Neutron diffraction data from an H/D-exchanged crambin crystal collected at the Protein Crystallography Station (PCS) showed diffraction beyond 1.1 Å resolution. This is the highest resolution neutron diffraction reported to date for a protein crystal and will reveal important details of the anisotropic motions of H and D atoms in protein structures.

Neutron protein crystallography reveals unique details of clinical drug binding to a human target enzyme

Volume 24 • Number 4 • 2013 Neutron News 20 Background Hydrogens (H) in structural biology – detection and function About half the atoms in proteins consist of hydrogen (H), with the other half being a combination of mostly C, N, O, and some S. In addition to these lighter atoms, metalloproteins also contain small amounts of bioactive metals, such as Mg, Ca, Zn, Mn, Fe, and a few others. Despite being the most abundant atom in proteins, H is the most diffi cult to observe in conventional X-ray crystal structures, due to their very small X-ray scattering length. H atoms are very important to protein folding, hydrogen bonding interactions, substrate or inhibitor binding, enzyme catalysis, and protein solvation [1]. Enzymes can also use H2O, OH–, H+, and H3O + to mediate catalysis but distinguishing between these species is very challenging using traditional methods [2, 3]. H is typically only defi nitively observed in X-ray structures at ultra high resolution (better than 1 Å) and even then, only a fraction of the total is observed. Seeing as ultra high-resolution structures only comprise ~0.7% of total deposited structures in the PDB (Protein Data Bank http://www.pdb.org), there is very little direct experimental information on H atom positions in proteins. Highlighting this, a recent study of the highest resolution neutron protein structure ever determined showed that the 1.1 Å resolution neutron structure directly showed ~95% of all H atoms, compared to only 30% in the 0.85 Å resolution X-ray structure [1]. Neutron protein crystallography offers unique information on H atoms and is highly complementary to X-ray scattering. From the atom types found in proteins, neutrons are scattered to a similar extent and offers isotope discrimination between H and deuterium (D), each with scattering lengths of –3.7 and 6.7 fm, respectively. To overcome signal cancellation and the large incoherent background from H scattering, it is routine to exchange all possible H atoms for its isotope D for neutron diffraction. This facilitates the direct observation of the protonation state of amino acid side chains, water orientation, H-bonding, and ligand binding [1, 2].