Uncertainty Quantification of a Seven Hole Pressure Probe

Wake bursting is a flowfield phenomena that can occur over the flaps of airplane wings. Wake bursting is not desired as it increases drag and decreases lift. An experiment took place in the University of Illinois low-speed low-turbulence wind tunnel that studied the effects of wake bursting over a multi-element airfoil. Data were collected by a seven-hole pressure probe in the wind tunnel by a small team of students. Angle of attack (α) and side slip angle (β) of the probe and velocity (v) of the air in the wind tunnel can be determined from measured data. My research assists a larger project on campus by aiding in uncertainty quantification of 7-hole probe data collected from the wind tunnel. In order to determine the accuracy of the data, methods were implemented to quantify uncertainty of 7-hole probe measurements. The jitter method was imbedded into an already existing MATLAB code that checked the calibrated data with the measured data. The overall uncertainties were 0.2857'° for α, 1.2032'° for β and 0.0481% for v It was determined that these uncertainties were small enough for the results to be valid.Ope

Preparation and characterization of highly thulium- and alumina-doped optical fibers for single-frequency fiber lasers

Thulium-doped fibers suitable for core-pumped single-frequency lasers were fabricated by the modified chemical vapor deposition (MCVD) method. Refractive index profile, doping profile and spectral absorption was measured. High doping concentration of thulium ions should be achieved to allow for high absorption of light at a pump wavelength while the thulium ions clustering should be avoided to prevent the cooperative upconversion and quenching processes. The fabricated fibers featured pump absorption up to 70dB/m at a pump wavelength of 1611nm. The single-frequency master oscillator with a resonator composed of a pair of fiber Bragg gratings and a thulium-doped fiber was demonstrated with predominantly single ended operation. We achieved a slope efficiency of 22% and a threshold of 22mW at a lasing wavelength of 1944nm

Dimeric and tetrameric forms of muscle fructose-1,6- bisphosphatase play different roles in the cell

Muscle fructose 1,6-bisphosphatase (FBP2), besides being a regulatory enzyme of glyconeogenesis also protects mitochondria against calcium stress and plays a key role in regulation of the cell cycle, promoting cardiomyocytes survival. However, in cancer cells, FBP2 acts as an anti-oncogenic/anti-proliferative protein. Here, we show that the physiological function of FBP2 depends both on its level of expression in a cell as well as its oligomerization state. Animal fructose-1,6-bisphosphatases are thought to function as tetramers. We present evidence that FBP2 exists in an equilibrium between tetramers and dimers. The dimeric form is fully active and insensitive to AMP, the main allosteric inhibitor of FBP2. Tetramerization induces the sensitivity of the protein to AMP, but it requires the presence of a hydrophobic central region in which leucine 190 plays a crucial role. Only the tetrameric form of FBP2 is retained in cardiomyocyte cell nucleus whereas only the dimeric form associates with mitochondria and protects them against stress stimuli, such as elevated calcium and H2O2 level. Remarkably, in hypoxic conditions, which are typical for many cancers, FBP2 ceases to interact with mitochondria and loses its pro-survival potential. Our results throw new light on the basis of the diverse role of FBP2 in cells

Structure of the interleukin-2 tyrosine kinase Src homology 2 domain; comparison between X-ray and NMR-derived structures

The crystal structure of the interleukin-2 tyrosine kinase Src homology domain (Itk SH2) is described and it is found that unlike in studies of this domain using NMR spectroscopy, cis-trans-prolyl isomerization is not readily detected in the crystal structure. Based on similarities between the Itk SH2 crystal form and the cis form of the Itk SH2 NMR structure, it is concluded that it is likely that the prolyl imide bond at least in part adopts the cis conformation in the crystal form. However, the lack of high-resolution data and the dynamic nature of the proline-containing loop mean that the precise imide-bond conformation cannot be determined and prolyl cis-trans isomerization in the crystal cannot be ruled out. Given the preponderance of structures that have been solved by X-ray crystallography in the Protein Data Bank, this result supports the notion that prolyl isomerization in folded proteins has been underestimated among known structures. Interestingly, while the precise status of the proline residue is ambiguous, Itk SH2 crystallizes as a domain-swapped dimer. The domain-swapped structure of Itk SH2 is similar to the domain-swapped SH2 domains of Grb2 and Nck, with domain swapping occurring at the β-meander region of all three SH2 domains. Thus, for Itk SH2 structural analysis by NMR spectroscopy and X-ray crystallography revealed very different structural features: proline isomerization versus domain-swapped dimerization, respectively

Hybrid Tetramers of Porcine Liver Fructose-1,6-bisphosphatase Reveal Multiple Pathways of Allosteric Inhibition

Fructose-1,6-bisphosphatase is a square planar tetramer of identical subunits, which exhibits cooperative allosteric inhibition of catalysis by AMP. Protocols for in vitrosubunit exchange provide three of five possible hybrid tetramers of fructose-1,6-bisphosphatase in high purity. The two hybrid types with different subunits in the top and bottom halves of the tetramer co-purify. Hybrid tetramers, formed from subunits unable to bind AMP and subunits with wild-type properties, differ from the wild-type enzyme only in regard to their properties of AMP inhibition. Hybrid tetramers exhibit cooperative, potent, and complete (100%) AMP inhibition if at least one functional AMP binding site exists in the top and bottom halves of the tetramer. Furthermore, titrations of hybrid tetramers with AMP, monitored by a tryptophan reporter group, reveal cooperativity and fluorescence changes consistent with an R- to T-state transition, provided that again at least one functional AMP site exists in the top and bottom halves of the tetramer. In contrast, hybrid tetramers, which have functional AMP binding sites in only one half (top/bottom), exhibit an R- to T-state transition and complete AMP inhibition, but without cooperativity. Evidently, two pathways of allosteric inhibition of fructose-1,6-bisphosphatase are possible, only one of which is cooperative

Origin of Cooperativity in the Activation of Fructose-1,6-bisphosphatase by Mg2+

Fructose-1,6-bisphosphatase requires a divalent metal cation for catalysis, Mg2+being its most studied activator. Phosphatase activity increases sigmoidally with the concentration of Mg2+, but the mechanistic basis for such cooperativity is unknown. Bound magnesium cations can interact within a single subunit or between different subunits of the enzyme tetramer. Mutations of Asp118, Asp121, or Glu97 to alanine inactivate the recombinant porcine enzyme. These residues bind directly to magnesium cations at the active site. Three different hybrid tetramers of fructose-1,6-bisphosphatase, composed of one wild-type subunit and three subunits bearing one of the mutations above, exhibit kinetic parameters (Km for fructose-1,6-bisphosphate, 1.1–1.8 μM; Ka for Mg2+, 0.34–0.76 mM; Ki for fructose-2,6-bisphosphate, 0.11–0.61 μM; and IC50 for AMP, 3.8–7.4 μM) nearly identical to those of the wild-type enzyme. Notwithstanding these similarities, thekcat parameter for each hybrid tetramer is approximately one-fourth of that for the wild-type enzyme. Evidently, each subunit in the wild-type tetramer can independently achieve maximum velocity when activated by Mg2+. Moreover, the activities of the three hybrid tetramers vary sigmoidally with the concentration of Mg2+ (Hill coefficients of ∼2). The findings above are fully consistent with a mechanism of cooperativity that arises from within a single subunit of fructose-1,6-bisphosphatase

Terpenoid synthase structures: a so far incomplete view of complex catalysis

The complexity of terpenoid natural products has drawn significant interest, particularly since their common (poly)isoprenyl origins were discovered. Notably, much of this complexity is derived from the highly variable cyclized and/or rearranged nature of the observed hydrocarbon skeletal structures. Indeed, at least in some cases it is difficult to immediately recognize their derivation from poly-isoprenyl precursors. Nevertheless, these diverse structures are formed by sequential elongation to acyclic precursors, most often with subsequent cyclization and/or rearrangement. Strikingly, the reactions used to assemble and diversify terpenoid backbones share a common carbocationic driven mechanism, although the means by which the initial carbocation is generated does vary. High-resolution crystal structures have been obtained for at least representative examples from each of the various types of enzymes involved in producing terpenoid hydrocarbon backbones. However, while this has certainly led to some insights into the enzymatic structure–function relationships underlying the elongation and simpler cyclization reactions, our understanding of the more complex cyclization and/or rearrangement reactions remains limited. Accordingly, selected examples are discussed here to demonstrate our current understanding, its limits, and potential ways forward

The N-terminal Segment of Recombinant Porcine Fructose-1,6-bisphosphatase Participates in the Allosteric Regulation of Catalysis

Residues 1–10 of porcine fructose-1,6-bisphosphatase (FBPase) are poorly ordered or are in different conformations, sensitive to the state of ligation of the enzyme. Deletion of the first 10 residues of FBPase reducesk cat by 30-fold and Mg2+ affinity by 20-fold and eliminates cooperativity in Mg2+ activation. Although a fluorescent analogue of AMP binds with high affinity to the truncated enzyme, AMP itself potently inhibits only 50% of the enzyme activity. Additional inhibition occurs only when the concentration of AMP exceeds 10 mM. Deletion of the first seven residues reduces k cat and Mg2+ affinity significantly but has no effect on AMP inhibition. The mutation of Asp9 to alanine reproduces the weakened affinity for Mg2+ observed in the deletion mutants, and the mutation of Ile10 to aspartate reproduces the AMP inhibition of the 10-residue deletion mutant. Changes in the relative stability of the known conformational states for loop 52–72, in response to changes in the quaternary structure of FBPase, can account for the phenomena above. Some aspects of the proposed model may be relevant to all forms of FBPase, including the thioredoxin-regulated FBPase from the chloroplast