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J Struct Biol. 2010 Jul;171(1):82-7. doi: 10.1016/j.jsb.2010.02.005. Epub 2010 Feb 10.

Fluorescence Detection of Heavy Atom Labeling (FD-HAL): a rapid method for identifying covalently modified cysteine residues by phasing atoms.

Author information

1
Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.

Abstract

Membrane protein crystallography frequently stalls at the phase determination stage due to poor crystal diffraction and the inability to identify heavy atom derivatization prior to data collection. Thus, a majority of time, effort and resources are invested preparing potential derivatized crystals for synchrotron data collection and analysis without knowledge of heavy atom labeling. To remove this uncertainty, we introduce Fluorescence Detection of Heavy Atom Labeling (FD-HAL) using tetramethylrhodamine-5-maleimide (a fluorescent maleimide compound) to monitor in-gel cysteine residue accessibility and ascertain covalent modification by mercury, platinum and gold compounds. We have tested this technique on three integral membrane proteins (LacY, vSGLT and mVDAC1) and can quickly assess the optimal concentrations, time and heavy atom compound to derivatize free cysteine residues in order to facilitate crystal phasing. This, in conjunction with cysteine scanning for incorporating heavy atoms at strategic positions, is a useful tool that will considerably assist in phasing membrane protein structures.

PMID:
20152903
DOI:
10.1016/j.jsb.2010.02.005
[Indexed for MEDLINE]

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