Buz Barstow

Evidence for liquid water during the high-density to low-density amorphous ice transition

Polymorphism of water has been extensively studied, but controversy still exists over the phase transition between high-density amorphous (HDA) and low-density amorphous (LDA) ice. We report the phase behavior of HDA ice inside high-pressure cryocooled protein crystals. Using X-ray diffraction, we demonstrate that the intermediate states in the temperature range from 80 to 170 K can be reconstructed as a linear combination of HDA and LDA ice, suggesting a first-order transition. We found evidence for a liquid state of water during the ice transition based on the protein crystallographic data. These observations open the possibility that the HDA ice induced by high-pressure cryocooling is a genuine glassy form of high-density liquid.

Alteration of citrine structure by hydrostatic pressure explains the accompanying spectral shift

A protein molecule is an intricate system whose function is highly sensitive to small external perturbations. However, no examples that correlate protein function with progressive subangstrom structural perturbations have thus far been presented. To elucidate this relationship, we have investigated a fluorescent protein, citrine, as a model system under high-pressure perturbation. The protein has been compressed to produce deformations of its chromophore by applying a high-pressure cryocooling technique. A closely spaced series of x-ray crystallographic structures reveals that the chromophore undergoes a progressive deformation of up to 0.8 Å at an applied pressure of 500 MPa. It is experimentally demonstrated that the structural motion is directly correlated with the progressive fluorescence shift of citrine from yellow to green under these conditions. This protein is therefore highly sensitive to subangstrom deformations and its function must be understood at the subangstrom level. These results have significant implications for protein function prediction and biomolecule design and engineering, because they suggest methods to tune protein function by modification of the protein scaffold.

Structural and Thermodynamic Characterization of T4 Lysozyme Mutants and the Contribution of Internal Cavities to Pressure Denaturation

Using small-angle X-ray scattering (SAXS) and tryptophan fluorescence spectroscopy, we have identified multiple compact denatured states of a series of T4 lysozyme mutants that are stabilized by high pressures. Recent studies imply that the mechanism of pressure denaturation is the penetration of water into the protein rather than the transfer of hydrophobic residues into water. To investigate water penetration and the volume change associated with pressure denaturation, we studied the solution behavior of four T4 lysozyme mutants having different cavity volumes at low and neutral pH up to a pressure of 400 MPa (0.1 MPa = 0.9869 atm). At low pH, L99A T4 lysozyme expanded from a compact folded state to a partially unfolded state with a corresponding change in radius of gyration from 17 to 32 A. The volume change upon denaturation correlated well with the total cavity volume, indicating that all of the molecules major cavities are hydrated with pressure. As a direct comparison to high-pressure crystal structures of L99A T4 lysozyme solved at neutral pH [Collins, M. D., Hummer, G., Quillin, M. L., Matthews, B. W., and Gruner, S. M. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 16668-16671], pressure denaturation of L99A and the structurally similar L99G/E108V mutant was studied at neutral pH. The pressure-denatured state at neutral pH is even more compact than at low pH, and the small volume changes associated with denaturation suggest that the preferential filling of large cavities is responsible for the compactness of the pressure-denatured state. These results confirm that pressure denaturation is characteristically distinct from thermal or chemical denaturation.

Coupling of Pressure-Induced Structural Shifts to Spectral Changes in a Yellow Fluorescent Protein

X-ray diffraction analysis of pressure-induced structural changes in the Aequorea yellow fluorescent protein Citrine reveals the structural basis for the continuous fluorescence peak shift from yellow to green that is observed on pressurization. This fluorescence peak shift is caused by a reorientation of the two elements of the Citrine chromophore. This study describes the structural linkages in Citrine that are responsible for the local reorientation of the chromophore. The deformation of the Citrine chromophore is actuated by the differential motion of two clusters of atoms that compose the beta-barrel scaffold of the molecule, resulting in a slight bending of the beta-barrel. The high-pressure structures also show a perturbation of the hydrogen bonding network that stabilizes the excited state of the Citrine chromophore. The perturbation of this network is implicated in the reduction of fluorescence intensity of Citrine. The blue-shift of the Citrine fluorescence spectrum resulting from the bending of the beta-barrel provides structural insight into the transient blue-shifting of isolated yellow fluorescent protein molecules under ambient conditions and suggests mechanisms to alter the time-dependent behavior of Citrine under ambient conditions.

The Cornell ERL prototype project

Synchrotron light sources based on Energy Recovery Linacs (ERLs) show promise to deliver X-ray beams with both brilliance and X-ray pulse duration far superior to the values that can be achieved with storage ring technology. Cornell University, in collaboration with Jefferson Laboratory, has proposed the construction of a prototype ERL. This 100MeV, 100mA CW superconducting electron accelerator will be used to study and resolve the many accelerator physics and technology issues of this type of machine. These studies are essential before ERLs can be confidently proposed for large-scale applications such as synchrotron light sources. Key issues include the generation of high average current, high brightness electron beams; acceleration and transport of these beams while preserving their brightness; adequate damping of higher order modes (HOMs) to assure beam stability; removal of large amounts of HOM power from the cryogenic environment; stable RF control of cavities operating at very high external Q; reduction of beam losses to very low levels; and the development of precision non-intercepting diagnostics to allow beam setup, control and characterization. Our prototype design allows us to address these and other issues over a broad range of parameter space. This design, along with recent progress on understanding these issues, will be presented.

First studies for a low temperature higher-order-mode absorber for the Cornell ERL prototype

Cornell University, in collaboration with Jefferson Laboratory, has proposed the construction of a prototype energy-recovery linac (ERL) to study the energy recovery concept with high current, low emittance beams. The beam with a current of up to 100 mA will excite significant higher-order-mode (HOM) power in the superconducting (s.c.) RF cavities with frequencies up to 100 GHz. Strong damping of the HOMs is essential for beam stability and to reduce the HOM losses to a few hundred Watts per meter. To achieve this demanding goal we plan to place RF absorbing material in the beam tubes between the cavities in the linac. However, this will require operating the HOM absorbers at temperatures below 80 K to simplify the thermal transition to the cavities at 2 K with low static losses to 2 K. One possible material candidate is ferrite, as it is used at room temperature in the HOM absorbers in the s.c. CESR cavities. In this paper we present experiments performed to study the RF absorption properties of ferrite at cryogenic temperatures in the frequency range from 1 GHz to 15 GHz. First results are shown and the resulting HOM damping is evaluated and discussed.

Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.

Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.

A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism

BackgroundFeFe-hydrogenases are the most active class of H2-producing enzymes known in nature and may have important applications in clean H2 energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O2.ResultsWe have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O2 levels eliminate growth. This pathway forms the basis for a genetic selection for O2 tolerance. Genetically selected hydrogenases did not show improved stability in O2 and in many cases had lost H2 production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer.ConclusionsRational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic H2 metabolism. Our results also indicate a H2-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H2-activating catalysts.

Overview of the Cornell ERL injector cryomodule

The Laboratory for Elementary-Particle Physics, Cornell University, in collaboration with Jefferson Lab is exploring the potential of a Synchrotron Radiation User Facility based on a multi-GeV, low emittance, Energy-Recovery Linac (ERL) with a 100 mA CW beam. The ERL injector will accelerate bunches from the electron source from 0.5 MeV to 5 MeV with minimal emittance growth. The injector and main linac of the ERL will be based on superconducting RF technology to provide CW operation. There will be one cryomodule with five 1300 MHz 2-cell cavities, each providing one MV of acceleration, corresponding to an accelerating field of about 4.3 MV/m in CW operation. Besides standard features such as an integrated helium vessel and mechanical tuner, each cavity has two input couplers, symmetrically placed on the beam pipe to cancel kicks due to coupler fields. For a 100 mA maximum injected beam current, each coupler must deliver 50 kW of beam power leading to a Qext of 4.6 /spl times/ 10/sup 4/ for matched beam loading conditions. Antenna- and loop-based HOM couplers can disturb beam emittance through kicks. We plan to avoid the use of such couplers. Following the strategy for B-factory SRF cavities, the beam pipe aperture has been enlarged on one side to propagate all higher order modes out to symmetric ferrite beam pipe loads. These are positioned outside the helium vessel and cooled to liquid nitrogen temperature. Ferrite properties at 77 K have been measured and the corresponding damping evaluated. To explore the full capabilities of the injector, energy gains up to 3 MV per cavity will be considered at lower beam currents. For this flexibility, the input coupling needs to be adjustable by a factor of 9.

Rapid curation of gene disruption collections using Knockout Sudoku

Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as 3 weeks of dedicated labor and at a cost of ∼