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B Leif Hanson

Publications and source records attributed to B Leif Hanson.

7 recordsLinked to original sources
PubMed2004

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.

Aldose-Ketose Isomerases↗
PubMed2003

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.

Cold Temperature↗
PubMed2002

Experiments testing the abatement of radiation damage in D-xylose isomerase crystals with cryogenic helium.

Helium is a more efficient cryogen than nitrogen, and for macromolecular data collection at high-flux beamlines will deliver lower temperatures. An open-flow helium cryostat developed at the University of Toledo (the Pinkerton Device) has been used for macromolecular data collection. This device differs from standard commercial He cryostats by having a much narrower aperture providing a high velocity stream of He around the crystal that maximizes convective and conductive heat exchange between the crystal and the cryogen. This paper details a series of experiments conducted at the IMCA-CAT 17ID beamline using one crystal for each experimental condition to examine whether helium at 16 K provided better radiation-damage abatement compared with nitrogen at 100 K. These studies used matched high-quality crystals (0.94 A diffraction resolution) of D-xylose isomerase derived from the commercial material Gensweet SGI. Comparisons show that helium indeed abates the indicators of radiation damage, in this case resulting in longer crystal diffractive lifetimes. The overall trend suggests that crystals maintain order and that high-resolution data are less affected by increased radiation load when crystals are cooled with He rather than N(2). This is probably the result of a lower effective temperature at the crystal with concomitant reduction in free-radical diffusion. Other features, such as an apparent phase transition in macromolecular crystals at lower temperatures, require investigation to broaden the utility of He use.

Aldose-Ketose Isomerases↗
PubMedDate not supplied

Mcg in 2030: new techniques for atomic position determination of immune complexes.

The lambda-type light chain dimer from a patient (Mcg) with multiple myeloma and amyloidosis was a pioneer protein for determining the three-dimensional structures of immunoglobulins, understanding the effects of ligand binding, and exploring the use of combinatorial methods to identify novel peptides complementary to protein active sites. Despite 30 years of intense study, there are still unanswered questions about the structure of the Mcg dimer, especially with respect to positions of hydrogen atoms and solvent molecules. In the present report, we describe two techniques that will help define the roles of solvent in ligand interactions and complex formation with this immunoglobulin fragment: (1) introduction of helium as a cryogenic agent during X-ray data collection; and (2) addition of neutron diffraction analyses. These techniques should provide improved resolution, and a more accurate structure of the Mcg dimer. Resolution enhancements of 0.5 A have been achieved in preliminary experiments with cryogenic helium, as compared with the best X-ray diffraction data obtained previously. In the near future, neutron diffraction studies should produce the first hydrogen structure for the Mcg dimer and help elucidate the ligand preferences and amyloidogenic properties of this eminently useful protein.

Amyloidosis↗