Linked Life Data

12077430

Source:http://linkedlifedata.com/resource/pubmed/id/12077430

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PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
Pt 7
pubmed:dateCreated
2002-6-21
pubmed:abstractText
Lanthanides can contribute a large anomalous component to X-ray scattering when present and ordered in a target crystal. This large anomalous signal is a useful source of phase information in X-ray crystallographic studies of biological macromolecules. Thiol-reactive lanthanide chelates were tested as a means of incorporation of lanthanides into protein crystals. Two compounds, each capable of being loaded with a lanthanide of choice, were synthesized: diethylenetriaminepentaacetic 3-(2-pyridyldithio)propionyl hydrazide (DTPA-PDPH) and 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic 3-(2-pyridyldithio)propionyl hydrazide (DOTA-PDPH). A cysteine mutant of the 34 kDa phosphate-binding protein (PBP-A197C) from Escherichia coli was used as a test case. PBP-A197C was labeled with DTPA-PDPH loaded with dysprosium. Characteristics of DTPA-PDPH enabled spectroscopic monitoring of the labeling reaction. Complete labeling of PBP-A197C was confirmed by mass spectrometry and SDS-PAGE analysis. Labeled PBP-A197C (PBP-A197C-DTPA-Dy) crystallized identically to unlabeled protein. X-ray diffraction data were collected from PBP-A197C-DTPA-Dy crystals in-house with a Cu Kalpha rotating-anode source and with a tuneable synchrotron source (ALS 5.0.2). Synchrotron data were collected at energies corresponding to the Dy L(III) edge f" peak and a high-energy remote. Each data set was treated as an independent SAD experiment. A large anomalous signal was present in the data collected in-house and at the synchrotron. The Dy site was easily located in anomalous difference Patterson maps calculated from each of the data sets. In each case, SAD phasing resulted in high-quality electron-density maps, as evidenced by the success of automated model building. The generality of the method was analyzed with several other test proteins. Labeling of some of these proteins with thiol-reactive lanthanide chelates was deleterious to protein solubility or crystallization. In two of the cases the lanthanide chelate was disordered in the crystals. These results suggest that this method may not be well suited for high-throughput crystallography. However, for difficult cases requiring a large anomalous signal, thiol-reactive lanthanide chelates may prove to be a valuable tool.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0907-4449
pubmed:author
pubmed:issnType
Print
pubmed:volume
58
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1111-7
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Thiol-reactive lanthanide chelates for phasing protein X-ray diffraction data.
pubmed:affiliation
Interdisciplinary Program in Biophysics, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville 22908, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.