Fluorescence Detection of Heavy Atom Labeling (FD-HAL): a rapid method for identifying covalently modified cysteine residues by phasing atoms.
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.
Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
Vincent
V
Chaptal
Rachna
R
Ujwal
Yiling
Y
Nie
Akira
A
Watanabe
Seunghyug
S
Kwon
Jeff
J
Abramson
D000818:Animals; D002623:Chemistry Techniques, Analytical; D003461:Crystallography; D003545:Cysteine / Q000737:chemistry*; D005453:Fluorescence; D006046:Gold / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry; D008628:Mercury / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D010984:Platinum / Q000737:chemistry; D051247:Sodium-Glucose Transport Proteins / Q000737:chemistry; D050995:Voltage-Dependent Anion Channel 1 / Q000737:chemistry
D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D051247:Sodium-Glucose Transport Proteins; C494763:Vdac1 protein, mouse; D010984:Platinum; D006046:Gold; C021499:lactose permease; D050995:Voltage-Dependent Anion Channel 1; D008628:Mercury; D003545:Cysteine