PMID- 12077430
OWN - NLM
STAT- MEDLINE
DCOM- 20030211
LR  - 20190605
IS  - 0907-4449 (Print)
IS  - 0907-4449 (Linking)
VI  - 58
IP  - Pt 7
DP  - 2002 Jul
TI  - Thiol-reactive lanthanide chelates for phasing protein X-ray diffraction data.
PG  - 1111-7
AB  - 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.
FAU - Purdy, Michael D
AU  - Purdy MD
AD  - Interdisciplinary Program in Biophysics, University of Virginia, 1300 Jefferson 
      Park Avenue, Charlottesville 22908, USA.
FAU - Ge, Pinghua
AU  - Ge P
FAU - Chen, Jiyan
AU  - Chen J
FAU - Selvin, Paul R
AU  - Selvin PR
FAU - Wiener, Michael C
AU  - Wiener MC
LA  - eng
GR  - AR44420/AR/NIAMS NIH HHS/United States
GR  - GM56251/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Research Support, U.S. Gov't, P.H.S.
DEP - 20020620
PL  - United States
TA  - Acta Crystallogr D Biol Crystallogr
JT  - Acta crystallographica. Section D, Biological crystallography
JID - 9305878
RN  - 0 (Chelating Agents)
RN  - 0 (Lanthanoid Series Elements)
RN  - 789U1901C5 (Copper)
RN  - 7A314HQM0I (Pentetic Acid)
SB  - IM
MH  - Chelating Agents/*chemistry
MH  - Copper/chemistry
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Escherichia coli/metabolism
MH  - Lanthanoid Series Elements/*chemistry
MH  - Models, Chemical
MH  - Models, Molecular
MH  - Pentetic Acid/chemistry
MH  - Plasmids/metabolism
MH  - Spectrometry, Fluorescence
MH  - Time Factors
MH  - X-Ray Diffraction/*instrumentation/*methods
EDAT- 2002/06/22 10:00
MHDA- 2003/02/13 04:00
CRDT- 2002/06/22 10:00
PHST- 2002/01/01 00:00 [received]
PHST- 2002/04/11 00:00 [accepted]
PHST- 2002/06/22 10:00 [pubmed]
PHST- 2003/02/13 04:00 [medline]
PHST- 2002/06/22 10:00 [entrez]
AID - S0907444902006509 [pii]
AID - 10.1107/s0907444902006509 [doi]
PST - ppublish
SO  - Acta Crystallogr D Biol Crystallogr. 2002 Jul;58(Pt 7):1111-7. doi: 
      10.1107/s0907444902006509. Epub 2002 Jun 20.