In vivo measurement of intramolecular distances using genetically encoded reporters.
The function of membrane proteins occurs in the context of the cell membrane in living cells acting in concert with various cell components such as other proteins, cofactors, etc. The understanding of the function at the molecular level requires structural techniques, but high resolution structural studies are normally obtained in vitro and in artificial membranes or detergent. Ideally the correlation of structure and function should be carried out in the native environment but most of the techniques applicable in vivo lack the high resolution necessary to track conformational changes on a molecular level. Here we report on the successful application of an improved variant of lanthanide-based resonance energy transfer a fluorescent based technique, to Shaker potassium channels expressed in live Xenopus oocytes. Lanthanide-based resonance energy transfer is particularly suitable to measure intramolecular distances with high resolution. The improvements reported in this work are mainly based on the use of two different small genetically encoded tags (the Lanthanide Binding Tag and the hexa-histidine tag), which due to their small size can be encoded at will in many positions of interest without distorting the protein's function. The technique reported here has the additional improvement that the two tags can be placed independently in contrast to previously described techniques that rely on chemical labeling procedures of thiols.
Sandtner
Walter
W
Department of Pediatrics, Institute for Molecular Pediatric Sciences, Gordon Center for Integrative Science, University of Chicago, Chicago, Illinois 60637, USA.
Bezanilla
Francisco
F
Correa
Ana M
AM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D006639:Histidine / Q000737:chemistry / Q000235:genetics*; D028581:Lanthanoid Series Elements / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D013194:Staining and Labeling; D014982:Xenopus laevis
D028581:Lanthanoid Series Elements; D010455:Peptides; C033223:polyhistidine; D006639:Histidine