Screening of nucleation conditions using levitated drops for protein crystallization
(2003) In Analytical Chemistry 75(7). p.1733-1740- Abstract
- The growth of suitable protein crystals is an essential step in the structure determination of a protein by X-ray crystallography. At present, crystals are mostly grown using trial-and-error procedures, and protocols that rapidly screen for the crystal nucleation step are rare. Presented here is an approach to minimize the consumption of precious protein material while searching for the nucleation conditions. Acoustically levitated drops of known protein concentration (0.25-1.5-L volumes) are injected with crystallizing agents using piezoelectric flow-through dispensers (ejecting 50-100-pL droplets at 1-9000 droplets/s). A restricted number of crystallizing agents representing three classes are used: poly(ethylene glycol), salts, and the... (More)
- The growth of suitable protein crystals is an essential step in the structure determination of a protein by X-ray crystallography. At present, crystals are mostly grown using trial-and-error procedures, and protocols that rapidly screen for the crystal nucleation step are rare. Presented here is an approach to minimize the consumption of precious protein material while searching for the nucleation conditions. Acoustically levitated drops of known protein concentration (0.25-1.5-L volumes) are injected with crystallizing agents using piezoelectric flow-through dispensers (ejecting 50-100-pL droplets at 1-9000 droplets/s). A restricted number of crystallizing agents representing three classes are used: poly(ethylene glycol), salts, and the viscous alcohol 2-methyl 2,4-pentanediol. From a digitized picture of the levitated drop volume, calculations are performed giving the concentrations of all components in the drop at any time during a "precipitation experiment". Supersaturation is the prerequisite for crystal nucleation, and protein precipitation indicates high supersaturation. A light source illuminates the levitated drop, and protein precipitation is monitored using right-angle light scattering. On the basis of these intensity measurements and the volume determination, precipitation diagrams for each crystallizing agent are constructed that give the protein/crystallizing agent concentration boundaries between the minimum and the maximum detectable protein precipitation. Guided by the concentration values obtained from such plots, when approaching the supersaturation region, separate crystallization drops are mixed and allowed to equilibrate under paraffin oil. At conditions in which microcrystals can be observed, the nucleation tendency of the macromolecule is confirmed. Optimization of crystallization conditions can then follow. Proteins tested include alcohol dehydrogenase and D-serine dehydratase. Alcohol dehydrogenase, known to crystallize easily, was used to evaluate whether the ultrasonic field inhibits nucleation. Details are given for the screening procedure of D-serine dehydratase, an enzyme earlier found to be difficult to crystallize reproducibly. The time and material-saving qualities of this method are emphasized, since a range of conditions can quickly be screened using small amounts of protein to roughly determine solubility characteristics of a protein before crystallization trials are initiated. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/132738
- author
- Santesson, Sabina LU ; Cedergren, Eila LU ; Johansson, Thomas ; Laurell, Thomas LU ; Nilsson, Johan LU and Nilsson, Staffan LU
- organization
- publishing date
- 2003
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Analytical Chemistry
- volume
- 75
- issue
- 7
- pages
- 1733 - 1740
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- pmid:12705610
- wos:000181993600034
- scopus:0242417548
- ISSN
- 1520-6882
- DOI
- 10.1021/ac020496y
- language
- English
- LU publication?
- yes
- additional info
- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Analytical Chemistry (S/LTH) (011001004), Biomedical Engineering (011200011), Biochemistry and Structural Biology (S) (000006142), Atomic physics (011013005)
- id
- 6700d9ee-3611-476b-ae79-72077658800f (old id 132738)
- date added to LUP
- 2016-04-01 11:36:57
- date last changed
- 2022-01-26 07:43:00
@article{6700d9ee-3611-476b-ae79-72077658800f,
abstract = {{The growth of suitable protein crystals is an essential step in the structure determination of a protein by X-ray crystallography. At present, crystals are mostly grown using trial-and-error procedures, and protocols that rapidly screen for the crystal nucleation step are rare. Presented here is an approach to minimize the consumption of precious protein material while searching for the nucleation conditions. Acoustically levitated drops of known protein concentration (0.25-1.5-L volumes) are injected with crystallizing agents using piezoelectric flow-through dispensers (ejecting 50-100-pL droplets at 1-9000 droplets/s). A restricted number of crystallizing agents representing three classes are used: poly(ethylene glycol), salts, and the viscous alcohol 2-methyl 2,4-pentanediol. From a digitized picture of the levitated drop volume, calculations are performed giving the concentrations of all components in the drop at any time during a "precipitation experiment". Supersaturation is the prerequisite for crystal nucleation, and protein precipitation indicates high supersaturation. A light source illuminates the levitated drop, and protein precipitation is monitored using right-angle light scattering. On the basis of these intensity measurements and the volume determination, precipitation diagrams for each crystallizing agent are constructed that give the protein/crystallizing agent concentration boundaries between the minimum and the maximum detectable protein precipitation. Guided by the concentration values obtained from such plots, when approaching the supersaturation region, separate crystallization drops are mixed and allowed to equilibrate under paraffin oil. At conditions in which microcrystals can be observed, the nucleation tendency of the macromolecule is confirmed. Optimization of crystallization conditions can then follow. Proteins tested include alcohol dehydrogenase and D-serine dehydratase. Alcohol dehydrogenase, known to crystallize easily, was used to evaluate whether the ultrasonic field inhibits nucleation. Details are given for the screening procedure of D-serine dehydratase, an enzyme earlier found to be difficult to crystallize reproducibly. The time and material-saving qualities of this method are emphasized, since a range of conditions can quickly be screened using small amounts of protein to roughly determine solubility characteristics of a protein before crystallization trials are initiated.}},
author = {{Santesson, Sabina and Cedergren, Eila and Johansson, Thomas and Laurell, Thomas and Nilsson, Johan and Nilsson, Staffan}},
issn = {{1520-6882}},
language = {{eng}},
number = {{7}},
pages = {{1733--1740}},
publisher = {{The American Chemical Society (ACS)}},
series = {{Analytical Chemistry}},
title = {{Screening of nucleation conditions using levitated drops for protein crystallization}},
url = {{https://lup.lub.lu.se/search/files/2561836/2374943.pdf}},
doi = {{10.1021/ac020496y}},
volume = {{75}},
year = {{2003}},
}