Crystallography Research Today is a free monthly online journal that collates and summarizes the latest research about Crystallography, including details on x-ray crystals, techniques, analyses, structures.

Hidden alternative structures of proline isomerase essential for catalysis.

Fraser JS, Clarkson MW, Degnan SC, Erion R, Kern D, Alber T

Department of Molecular and Cell Biology/QB3, University of California, Berkeley, California 94720-3220, USA.

A long-standing challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis. X-ray crystallography can provide snapshots of conformational substates sampled during enzymatic reactions, while NMR relaxation methods reveal the rates of interconversion between substates and the corresponding relative populations. However, these current methods cannot simultaneously reveal the detailed atomic structures of the rare states and rationalize the finding that intrinsic motions in the free enzyme occur on a timescale similar to the catalytic turnover rate. Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CYPA, also known as PPIA). A conservative mutation outside the active site was designed to stabilize features of the previously hidden minor conformation. This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate. These studies introduce crystallographic approaches to define functional minor protein conformations and, in combination with NMR analysis of the enzyme dynamics in solution, show how collective motions directly contribute to the catalytic power of an enzyme.

Published 3 December 2009 in Nature, 462(7273): 669-73.
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Articles on Crystallography published 25 November 2009:

Structural and genetic analysis of X-ray scattering by spores of Bacillus subtilis.   J Bacteriol, 191(24): 7620-2.

Dormant spores of Bacillus subtilis exhibit two prominent X-ray scattering peaks. These peaks persisted in spores lacking most alpha/beta-type small, acid-soluble protein or the CotE protein responsible for assembly of much spore coat protein, but they were absent from spores of strains lacking the late sporulation-specific transcription factor GerE. [Abstract] [Full-text]

Articles on Crystallography published 16 November 2009:

Crystal structure and catalytic mechanism of 4-methylmuconolactone methylisomerase.   J Biol Chem, 284(47): 32709-16.

When methyl-substituted aromatic compounds are degraded via ortho (intradiol)-cleavage of 4-methylcatechol, the dead-end metabolite 4-methylmuconolactone (4-ML) is formed. Degradation of 4-ML has only been described in few bacterial species, including Pseudomonas reinekei MT1. The isomerization of 4-ML to 3-methylmuconolactone (3-ML) is the first step required for the mineralization of 4-ML and is catalyzed by an enzyme termed 4-methylmuconolactone methylisomerase (MLMI). We identified the gene ... [Abstract] [Full-text]

Articles on Crystallography published 3 November 2009:

Triple-helix DNA structural studies using a Love wave acoustic biosensor.   Biosens Bioelectron, 25(4): 702-7.

The development of sensors for detecting the conformation of surface-attached molecules is an emerging field with significance in the pharmaceutical industry and in drug design. In this work, triplex-forming oligos (TFOs), a separate class of non-natural DNA bending agents that can affect the mechanical properties of DNA through the formation of triple-helical structures of specific conformation and/or flexibility, are used as a model system in combination with an acoustic biosensor to ... [Abstract] [Full-text]

Articles on Crystallography published 30 October 2009:

Internodal myelination during development quantitated using X-ray diffraction.   J Struct Biol, 168(3): 521-6.

Characterizing the formation, accretion, and stability of myelin during development, maturation, and senescence is important for better understanding critical periods in the function of the nervous system in normal growth and following environmental insult or genetic mutation. Although there are numerous studies on the ultrastructural, biochemical, and genetic aspects of myelin development and maturation, few have used X-ray diffraction (XRD), which can rapidly provide unique metrics about ... [Abstract] [Full-text]

Articles on Crystallography published 27 October 2009:

Single crystal structural and absorption spectral characterizations of nitric oxide synthase complexed with N(omega)-hydroxy-L-arginine and diatomic ligands.   Biochemistry, 48(43): 10246-54.

The X-ray structures of neuronal nitric oxide synthase (nNOS) with N(omega)-hydroxy-l-arginine (l-NHA) and CO (or NO) bound have been determined at 1.91-2.2 A resolution. Microspectrophotometric techniques confirmed reduced redox state and the status of diatomic ligand complexes during X-ray diffraction data collection. The structure of nNOS-NHA-NO, a close mimic to the dioxygen complex, provides a picture of the potential interactions between the heme-bound diatomic ligand, substrate l-NHA, ... [Abstract] [Full-text]

Articles on Crystallography published 19 October 2009:

High-throughput crystallography for structural genomics.   Curr Opin Struct Biol, 19(5): 573-84.

Protein X-ray crystallography recently celebrated its 50th anniversary. The structures of myoglobin and hemoglobin determined by Kendrew and Perutz provided the first glimpses into the complex protein architecture and chemistry. Since then, the field of structural molecular biology has experienced extraordinary progress and now more than 55000 protein structures have been deposited into the Protein Data Bank. In the past decade many advances in macromolecular crystallography have been driven by ... [Abstract] [Full-text]

Recent developments in phasing and structure refinement for macromolecular crystallography.   Curr Opin Struct Biol, 19(5): 566-72.

Central to crystallographic structure solution is obtaining accurate phases in order to build a molecular model, ultimately followed by refinement of that model to optimize its fit to the experimental diffraction data and prior chemical knowledge. Recent advances in phasing and model refinement and validation algorithms make it possible to arrive at better electron density maps and more accurate models. [Abstract] [Full-text]

Lipid-protein interactions probed by electron crystallography.   Curr Opin Struct Biol, 19(5): 560-5.

Electron crystallography is arguably the only electron cryomicroscopy (cryoEM) technique able to deliver an atomic-resolution structure of membrane proteins embedded in the lipid bilayer. In the electron crystallographic structures of the light driven ion pump, bacteriorhodopsin, and the water channel, aquaporin-0, sufficiently high resolution was obtained and both lipid and protein were visualized, modeled, and described in detail. An extensive network of lipid-protein interactions mimicking ... [Abstract] [Full-text]

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