[8] Using cosolvents to stabilize protein conformation for crystallization

Sousa, Rui

doi:10.1016/s0076-6879(97)76054-3

Cited by 9 publications

(3 citation statements)

References 67 publications

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“…Heras & Martin, 2005). This 'mother liquor' (see reviews by Sousa, 1997;Giegé & McPherson, 2001) or the cryo-buffer (normally constituted of the mother liquor mixed with a cryoprotectant agent such as glycerol) usually is partially retained around the crystal during its cooling and actual X-ray beam exposure. Thus, a very supportive and protective environment (in addition to the use of low-temperature-based protection and limited radiation doses) is provided for protein crystals being subjected to X-ray diffraction.…”

Section: New Technical Developments Might Provide Additional Reductiomentioning

confidence: 99%

Radiation damage to protein specimens from electron beam imaging and diffraction: a mini-review of anti-damage approaches, with special reference to synchrotron X-ray crystallography

Massover

2006

J Synchrotron Radiat

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Recent research progress using X-ray cryo-crystallography with the photon beams from third-generation synchrotron sources has resulted in recognition that this intense radiation commonly damages protein samples even when they are held at 100 K. Other structural biologists examining thin protein crystals or single particle specimens encounter similar radiation damage problems during electron diffraction and imaging, but have developed some effective countermeasures. The aim of this concise review is to examine whether analogous approaches can be utilized to alleviate the X-ray radiation damage problem in synchrotron macromolecular crystallography. The critical discussion of this question is preceded by presentation of background material on modern technical procedures with electron beam instruments using 300-400 kV accelerating voltage, low-dose exposures for data recording, and protection of protein specimens by cryogenic cooling; these practical approaches to dealing with electron radiation damage currently permit best resolution levels of 6 A (0.6 nm) for single particle specimens, and of 1.9 A for two-dimensional membrane protein crystals. Final determination of the potential effectiveness and practical value of using such new or unconventional ideas will necessitate showing, by experimental testing, that these produce significantly improved protection of three-dimensional protein crystals during synchrotron X-ray diffraction.

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Section: New Technical Developments Might Provide Additional Reductiomentioning

confidence: 99%

Radiation damage to protein specimens from electron beam imaging and diffraction: a mini-review of anti-damage approaches, with special reference to synchrotron X-ray crystallography

Massover

2006

J Synchrotron Radiat

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“…This¯exibility is a source of structural heterogeneity which may act to inhibit crystallization. Structural heterogeneity is particularly prevalent when one is dealing with multidomain proteins where the interdomain contacts may be rather¯exible (Sousa, 1997). It was thought that introduction of a ligand to the crystallization trials of FT domain might act to enhance crystal formation, particularly if such conformational¯exibility exists.…”

Section: Protein Puri®cation and Crystallizationmentioning

confidence: 99%

Crystallization and preliminary X-ray analysis of the formiminotransferase domain from the bifunctional enzyme formiminotransferase–cyclodeaminase

Kohls

Croteau

Mejia

et al. 1999

Acta Crystallogr D Biol Cryst

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) is a bifunctional enzyme involved in the histidine-degradation pathway which exhibits speci®city for polyglutamylated folate substrates. The ®rst function of the enzyme transfers the formimino group of formiminoglutamate to the N 5 position of tetrahydrofolate, while the second function catalyses the cyclodeamination of the formimino group, yielding N 5,10 -methenyl-tetrahydrofolate, with ef®cient channeling of the intermediate between these activities. Initial studies have shown that the enzyme consists of eight identical subunits of 62 kDa each, arranged as a circular tetramer of dimers. It is this formation which results in two different dimeric interfaces, which are necessary for the two different activities. The identical subunits have been shown to consist of two domains, each of which can be obtained as dimers. The formiminotransferase domain has been crystallized in the presence of the substrate analogue folinic acid. The crystals belong to space group P2 1 2 1 2 1 , with unit-cell dimensions a = 64.4, b = 103.7, c = 122.3 A Ê . Both a native data set and a mercurial derivative data set have been collected to 2.8 A Ê resolution.

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“…In Biochemistry, glycerol is widely used in protein isolation pathways, usually at concentrations 5-25 %, to facilitate folding and to increase protein solubility and stability (Sousa, 1997). Quality control studies of purified proteins include amino acid composition analysis, a classical, rather complex analytical technique, which is indispensable for protein quantification.…”

Section: Introductionmentioning

confidence: 99%

Glycerol affects the quantification of aspartate and glutamate in acid-hydrolyzed proteins

et al. 1998

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Glycerol is widely used in protein isolation pathways to improve folding and solubility of the proteins of interest. Amino acid composition analysis of protein samples hydrolyzed in the presence of glycerol resulted in underestimation of aspartate and glutamate, when compared to hydrolysis in the absence of glycerol. Quantification of free asparagine, aspartic acid, glutamine and glutamic acid hydrolyzed with hydrochloric acid or methanesulfonic acid in the presence of glycerol resulted in poor recoveries of aspartate and glutamate (between 6 and 66%). Gas chromatography-mass spectrometry analysis of the hydrolyzates revealed, as expected, the presence of esterification products. The esters were formed between the primary and secondary hydroxyl groups of the glycerol and both carboxyl groups of the amino acids. Protein samples intended for compositional analysis should be free of glycerol.

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[8] Using cosolvents to stabilize protein conformation for crystallization

Cited by 9 publications

References 67 publications

Radiation damage to protein specimens from electron beam imaging and diffraction: a mini-review of anti-damage approaches, with special reference to synchrotron X-ray crystallography

Radiation damage to protein specimens from electron beam imaging and diffraction: a mini-review of anti-damage approaches, with special reference to synchrotron X-ray crystallography

Crystallization and preliminary X-ray analysis of the formiminotransferase domain from the bifunctional enzyme formiminotransferase–cyclodeaminase

Glycerol affects the quantification of aspartate and glutamate in acid-hydrolyzed proteins

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