Local, atomic-level elastic strain measurements of metallic glass thin films by electron diffraction.

A novel technique is used to measure the atomic-level elastic strain tensor of amorphous materials by tracking geometric changes of the first diffuse ring of selected area electron diffraction patterns (SAD). An automatic procedure, which includes locating the centre and fitting an ellipse to the diffuse ring with sub-pixel precision is developed for extracting the 2-dimensional strain tensor from the SAD patterns. Using this technique, atomic-level principal strains from micrometre-sized regions of freestanding amorphous Ti0.45Al0.55 thin films were measured during in-situ TEM tensile deformation. The thin films were deformed using MEMS based testing stages that allow simultaneous measurement of the macroscopic stress and strain. The calculated atomic-level principal strains show a linear dependence on the applied stress, and good correspondence with the measured macroscopic strains. The calculated Poisson's ratio of 0.23 is reasonable for brittle metallic glasses. The technique yields a strain accuracy of about 1×10(-4) and shows the potential to obtain localized strain profiles/maps of amorphous thin film samples.

13C NMR studies of the enzyme-product complex of Bacillus subtilis chorismate mutase.

The chorismate mutase reaction is a rare enzyme-catalyzed 3,3-sigmatropic rearrangement of chorismate to prephenate. Bacillus subtilis chorismate mutase was overproduced and purified from Escherichia coli XL1-Blue (pBSCM2) using a modification of the procedure of Gray et al. (Gray, J. V., Grolinelli-Pimpaneau, B., & Knowles, J. R. (1990) Biochemistry 29, 376-383); the modification leads to minimal contaminating prephenate dehydratase activity (< 0.001%). The native molecular mass of B. subtilis chorismate mutase was determined by gel filtration to be approximately 44 kDa, indicative of a homotrimer of the 14.5-kDa subunits as determined by electrospray mass spectrometry. 13C NMR was used to study the structure of [U-13C]prephenate bound at the active site of B. subtilis chorismate mutase. All the enzyme-bound 13C NMR resonances of [U-13C]prephenate were assigned, and where possible, 1JC,Cs were quantified; [1,3,5,8-13C]prephenate and [2,6,9-13C]prephenate, prepared respectively from [1,3,5,8-13C]chorismate and [2,6,9-13C]chorismate, aided the 13C NMR resonance assignments. Enzyme-bound prephenate exhibits remarkably different chemical shifts relative to free prephenate; the chemical shift changes range from -6.6 ppm for the C6 resonance to 5.6 ppm for the C5 resonance, suggesting a strong perturbation of the C5-C6 bond. 13C NMR studies of model compounds at various pH values and in various solvents suggest that the observed 13C chemical shift changes of enzyme-bound prephenate cannot be rationalized solely on the basis of changes in the pKas of the carboxylic acid groups or hydrophobic solvation at the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase.

31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.

15N and 13C NMR studies of ligands bound to the 280,000-dalton protein porphobilinogen synthase elucidate the structures of enzyme-bound product and a Schiff base intermediate.

Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). Despite the 280,000-dalton size of PBGS, much can be learned about the reaction mechanism through 13C and 15N NMR. To our knowledge, these studies represent the largest protein complex for which individual nuclei have been characterized by 13C or 15N NMR. Here we extend our 13C NMR studies to PBGS complexes with [3,3-2H2,3-13C]ALA and report 15N NMR studies of [15N]ALA bound to PBGS. As in our previous 13C NMR studies, observation of enzyme-bound 15N-labeled species was facilitated by deuteration at nitrogens that are attached to slowly exchanging hydrogens. For holo-PBGS at neutral pH, the NMR spectra reflect the structure of the enzyme-bound product porphobilinogen (PBG), whose chemical shifts are uniformly consistent with deprotonation of the amino group whose solution pKa is 11. Despite this local environment, the protons of the amino group are in rapid exchange with solvent (kexchange greater than 10(2) s-1). For methyl methanethiosulfonate (MMTS) modified PBGS, the NMR spectra reflect the chemistry of an enzyme-bound Schiff base intermediate that is formed between C4 of ALA and an active-site lysine. The 13C chemical shift of [3,3-2H2,3-13C]ALA confirms that the Schiff base is an imine of E stereochemistry. By comparison to model imines formed between [15N]ALA and hydrazine or hydroxylamine, the 15N chemical shift of the enzyme-bound Schiff base suggests that the free amino group is an environment resembling partial deprotonation; again the protons are in rapid exchange with solvent. Deprotonation of the amino group would facilitate formation of a Schiff base between the amino group of the enzyme-bound Schiff base and C4 of the second ALA substrate. This is the first evidence supporting carbon-nitrogen bond formation as the initial site of interaction between the two substrate molecules.