Environment-sensitive Labels in Multiplex Fluorescence Analyses of Protein-DNA Complexes

Abstract

Fluorescein is widely used for protein labeling because of its high extinction coefficient and fluorescence emission quantum yield. However, its emission is readily quenched by various pathways. We exploit these properties of fluorescein to examine the self-association of a DNA binding protein and determine the amount of the protein in gel-shifted complexes with specific DNA. A construct ( The "gel mobility shift" or "band-shift" assay is widely used to detect and analyze protein-DNA interactions. The most direct way to determine the stoichiometry of protein-DNA complexes resolved in such experiments is to measure independently the amounts of both DNA and protein in a band on a gel. Quantitation of DNA in the shifted complex is relatively straightforward with 32 P end-labeling, but accurate measurement of protein can be quite challenging. Protein quantities in shifted complexes have been measured in double-labeling experiments using Coomassie staining, Western blot, and radioisotopic labeling of the protein with 3 H, 125 I, and 35 S (1-7). Drawbacks of these methods include limitations on sensitivity, difficulty in ensuring accurately the specific activity of radiolabeled proteins, and the need to handle the gel after electrophoresis for staining or blotting, or to cut out gel bands for scintillation counting. Consequently, few accurate determinations of the molar ratios of protein-to-DNA have been reported.We have shown that multiplex fluorescence analysis is a superior alternative method of characterizing protein-DNA complexes (8). In our earlier work, we described the first detailed analysis of the application of fluorescent polycationic intercalating dyes with high affinity for double-stranded DNA to the study of protein-DNA interactions. Such intercalating dyes, in conjunction with high sensitivity laser-excited, confocal, fluorescence scanning systems, allow detection of doublestranded DNA in agarose or acrylamide gels with a sensitivity comparable to that of autoradiography (9 -12). We used such dyes in gel mobility shift assays to detect the multiple protein-DNA complexes formed by the heat shock transcription factor (HSF) 1 (8,13,14). Using a truncation of Kluyveromyces lactis HSF that contains the DNA-binding and trimerization domains (HSF DT ), we were able to detect fluorescent dye-labeled HSF DT -DNA complexes with a spatial resolution superior to that of conventional autoradiography, and therefore we were able to analyze multimer protein-DNA complexes that are not resolved by traditional methods. An analysis of the mobilities of the multiple HSF DT -DNA complexes, indicated that HSF forms multimeric complexes on DNA by the addition of trimeric units (8).To measure the absolute quantities of protein and DNA in each complex, we developed a two-color mobility shift fluorescence assay with a mutant HSF DT engineered for site-specific labeling with fluorescein and target DNA labeled with thiazole orange-thiazole blue heterodimer (TOTAB), an "energy transfer" dye (15, 16). A technical difficulty was enco...

The GCN4 leucine zipper can functionally substitute for the heat shock transcription factor’s trimerization domain

“…The amount of D(21L)Z S-Fl in each complex was measured by comparing its¯uorescein emission to that of known amounts of native D(21L)Z S-Fl in microtiter wells. To observe any changes in thē uorescence of labeled protein caused by DNAbinding, we measured the¯uorescence of known amounts of D(21L)Z S-Fl incubated with molar excess amounts of DNA (Drees et al, 1996). Thē uorescence emission of D(21L)Z S-Fl did not appear to be changed by binding to its speci®c site on DNA (data not shown).…”

“…Direct stoichiometric determination using a two-color¯uorescent gel mobility shift assay con®rmed that the ®rst complex formed was a trimer (Rye et al, 1993;Drees et al, 1996). The larger, multimeric complexes were formed by addition of trimeric units, making hexamers, nonamers, etc.…”

“…Based on their electrophoretic mobilities in comparison to other complexes, these lower mobility complexes that are formed on a three-box site are likely to be two trimers or a hexamer. It is interesting that the HSFleucine zipper chimeras form multimeric complexes in a manner analogous to that of the HSF truncations, which forms multimers of trimers as protein concentration is increased (Rye et al, 1993;Drees et al, 1996).…”

“…Trimerization of the HSF truncation has an association constant of at least 10 À16 M 2 (Drees et al, 1996), and is mostly trimeric under the conditions used in the binding assays. The fact that D(21L)T has different af®nities for the two-box and three-box HSEs suggests that the presence of a third GAA box in the binding site increases HSF's binding af®nity by providing speci®c DNA contacts for all three trimer subunits.…”

Laser-induced hole filling by energy transfer between chromophores bound to DNA

“…orster theory predicts that the efficiency of energy transfer depends upon the inverse sixth power of distance. This character is particularly useful for a quantitative measurement of distances in the range of 20-80 ( A: This spectroscopic technique has been extensively applied to nucleic acid analysis such as DNA hybridization [9,10], DNA sequencing [11], sequence-directed structures [12,13] and DNA-protein interactions [14,15].…”