Production of crystals of human aldose reductase with very high resolution diffraction

Lamour, Valérie; Barth, Patrick; Rogniaux, Hélène; Poterszman, Arnaud; Howard, Eduardo; Mitschler, A.; Dorsselaer, Alain Van; Podjarny, A.; Moras, Dino

doi:10.1107/s0907444998013365

Cited by 39 publications

(46 citation statements)

References 9 publications

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“…example, human aldose reductase crystals have a V M of 2.10 Å 3 Da À1 , giving an estimated d PDB of $1.35 Å . This confirms that the value of 1.7 Å initially reported at a home source (Lamour et al, 1999) was below what might be obtained. At the same time, (10) does not predict that some aldose reductase crystals can diffract to 0.66 Å resolution (Howard et al, 2004).…”

Section: Possible Applicationssupporting

confidence: 66%

On the use of logarithmic scales for analysis of diffraction data

Urzhumtsev

Afonine

Adams

2009

Acta Crystallogr D Biol Crystallogr

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Predictions of the possible model parameterization and of the values of model characteristics such as R factors are important for macromolecular refinement and validation protocols. One of the key parameters defining these and other values is the resolution of the experimentally measured diffraction data. The higher the resolution, the larger the number of diffraction data N ref , the larger its ratio to the number N at of non-H atoms, the more parameters per atom can be used for modelling and the more precise and detailed a model can be obtained. The ratio N ref /N at was calculated for models deposited in the Protein Data Bank as a function of the resolution at which the structures were reported. The most frequent values for this distribution depend essentially linearly on resolution when the latter is expressed on a uniform logarithmic scale. This defines simple analytic formulae for the typical Matthews coefficient and for the typically allowed number of parameters per atom for crystals diffracting to a given resolution. This simple dependence makes it possible in many cases to estimate the expected resolution of the experimental data for a crystal with a given Matthews coefficient. When expressed using the same logarithmic scale, the most frequent values for R and R free factors and for their difference are also essentially linear across a large resolution range. The minimal R-factor values are practically constant at resolutions better than 3 Å , below which they begin to grow sharply. This simple dependence on the resolution allows the prediction of expected R-factor values for unknown structures and may be used to guide model refinement and validation.

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Section: Possible Applicationssupporting

confidence: 66%

On the use of logarithmic scales for analysis of diffraction data

Urzhumtsev

Afonine

Adams

2009

Acta Crystallogr D Biol Crystallogr

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“…The analysis of the electron density distribution of human aldose reductase is also underway. Crystals diffracting to very high resolution (Lamour et al, 1999) have been grown for this enzyme of 315 amino acids and diffraction data have been collected to 0.65 A Ê resolution at the APS synchrotron (Mitschler et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Refinement of proteins at subatomic resolution withMOPRO

et al. 2001

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Crystallography at subatomic resolution permits the observation and measurement of the non-spherical character of the atomic electron density. Charge density studies are being performed on molecules of increasing size. The MOPRO least-squares re®nement software has thus been developed, by extensive modi®cations of the program MOLLY, for protein and supramolecular chemistry applications. The computation times are long because of the large number of re¯ections and the complexity of the multipolar model of the atomic electron density; the structure factor and derivative calculations have thus been parallelized. Stereochemical and dynamical restraints as well as the conjugate gradient algorithm have been implemented. A large number of the normal matrix off-diagonal terms turn out to be very small and the block diagonal approximation is thus particularly ef®cient in the case of large structures at very high resolution.

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“…where E is the enzyme concentration, I is the inhibitor concentration, and K′ I is the apparent inhibition constant (36,37).…”

Section: -Chloro-n-(4-cyanobenzyl)-2-hydroxybenzaminementioning

confidence: 99%

“…B. hALR2 Expression and Purification. The expression and purification of hALR2 was conducted as described previously with several modifications (17,36). The gene for wild-type (WT) hALR2 was obtained from Dr. Alberto Podjarny in the pET-15b expression vector (Novagen) and sequenced.…”

Section: -Chloro-n-(4-cyanobenzyl)-2-hydroxybenzaminementioning

confidence: 99%

Electrostatic Fields Near the Active Site of Human Aldose Reductase: 1. New Inhibitors and Vibrational Stark Effect Measurements

2008

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Vibrational Stark effect spectroscopy was used to measure electrostatic fields in the hydrophobic region of the active site of human aldose reductase (hALR2). A new hALR2 inhibitor was designed and synthesized that contains a nitrile probe with a Stark tuning rate of 0.77 cm -1 /(MV/cm). Mutations to amino acid residues in the vicinity of the nitrile functional group were selected based on electrostatics calculations, possible complications from hydrogen bonds near the nitrile, and comparison with the active site of human aldehyde reductase, whose structure is very similar. Changes in the absorption energy of the nitrile probe when bound to those mutated proteins were then used to quantify perturbations to the protein's electrostatic field. Electrostatic field changes as large as -10 MV/cm were observed. Measured electrostatic fields were compared to predictions based on continuum electrostatics calculations, revealing that substantial modifications to the calculation strategy are necessary. The effects of hydrogen bonding of amino acid side chains to the nitrile probe are considered, and applications of vibrational Stark effect spectroscopy to investigations of ligand binding and biological function are discussed.The highly organized three-dimensional structure of a protein can support large variations in internal electrostatic fields that influence every aspect of the protein's function (1-3) including folding (4), chemical reactivity and kinetics (5-7), and protein-protein interactions (8). Measurements of the strength and direction of these fields are therefore of great importance, and represent a long-standing biophysical challenge (9-16). One strategy that has been developed in recent years is vibrational Stark effect (VSE 1 ) spectroscopy, which describes the influence of an electric field on a molecular vibrational frequency and can be observed through infrared spectroscopy (17,18). VSE spectroscopy is a twostep experiment. In the first step, a vibrational probe is selected and the effect of a known external applied electric field on the infrared absorption frequency is used to calibrate sensitivity of the oscillator's vibrational frequency to an electric field. The magnitude of this effect is called the Stark tuning rate, and is taken as an intrinsic property of the oscillator. In the second step, the calibrated vibrational probe is inserted into a protein of interest and used as a highly local, sensitive, and directional reporter of changes of the protein's electrostatic field as perturbations are made to the protein, for example by amino acid mutagenesis. We use the term "vibrational Stark effect" to describe both the effect of a known applied external electric field on an oscillator's vibrational frequency and the absorption frequency shifts in the IR spectrum of the calibrated spectator vibrational probe when it is exposed to a perturbation inside a protein.Here we introduce an ideal vibrational Stark probe, the nitrile (CtN) functional group (19), into the active site of the protein human...

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Production of crystals of human aldose reductase with very high resolution diffraction

Cited by 39 publications

References 9 publications

On the use of logarithmic scales for analysis of diffraction data

On the use of logarithmic scales for analysis of diffraction data

Refinement of proteins at subatomic resolution withMOPRO

Electrostatic Fields Near the Active Site of Human Aldose Reductase: 1. New Inhibitors and Vibrational Stark Effect Measurements

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