Rapid Kinetic Analysis of Protein Synthesis - Biophysical Approaches to Translational Control of Gene Expression - page 118

7.3 Fluorescence Labeling

For proteins, the most common approach is to introduce single cysteine residues,
preferably at non-conserved surface positions, and use thiol-reactive derivatives of
fluorophores to attach fluorescent groups at the desired positions. Intrinsic cysteines
in the protein to be labeled should be removed, unless they are buried and inacces-
sible to the labeling reagents. Another approach, which avoids mutagenesis, is to
introduce fluorescence labels in a random way by labeling lysine or cysteine residues
at the surface of the protein. Usually, there are many potentially reactive residues; to
avoid detrimental effects of extensive modification on the activity of the protein,
labeling should be limited to a few residues per molecule on the average. One poten-
tial disadvantage of the approach is that the labeling is likely to result in a heteroge-
neous pool of molecules with dyes attached at different positions which may report
different rearrangements, resulting in multiple, often poorly defined kinetic phases.
Alternatively, fluorescence tags can be introduced at the N- or C-terminus of the
proteins; however, the potential of such labels for monitoring conformational rear-
rangements along the reaction coordinate has been poorly explored.
RNA can be labeled in a number of ways, e.g., at the 5¢ or 3¢ ends or at internal
positions using fluorescent nucleotide derivatives, many of which are commercially
available. In the growing field of RNA chemistry, new methods emerge rapidly. An
exciting new possibility is to introduce small tags that specifically bind fluorophores,
such as the tetracysteine tag (Lumio tag). For labeling natural tRNA, modifications
provide a number of useful reactive groups, two of which, thioU at position 8
(Johnson et al. 1982 ) and dihydroU in the D loop of tRNA (Wintermeyer and Zachau
1974 ), are commonly found in bacterial tRNAs; an asp 3 U modi fi cation at position
47 of some tRNAs is often used as well. Ribosomes can be labeled in a variety of
ways. In a relatively simple approach, the ribosomes are labeled by supplying a
fluorescence-labeled ribosomal protein to ribosomes that lack the respective protein
because it was deleted from the chromosome in the bacterial strain used as a source
of ribosomes. So far, this approach has been successfully applied to proteins L1,
L11, and L29 (Munro et al. 2010 ; Seo et al. 2006 ). Ribosomal protein L12 can be
easily removed from the ribosomes by ethanol/salt treatment and replaced by the
purified fluorescence-labeled protein. More complicated labeling protocols suggest
full reconstitution of ribosomal subunits using purified components (Hickerson
et al. 2005 ), one of which is labeled, or use labeled oligonucleotides base-paired to
engineered extensions in rRNA (Dorywalska et al. 2005 ) .
Methods for fluorescence labeling are well-established and often can be
downloaded from the web-sites of the companies from which the dye is purchased.
However, an optimization of labeling conditions for each particular protein/RNA-
dye pair is recommended, as it may improve both yield and purity of labeled prod-
uct. Because labeling may alter the properties of the target macromolecules, the
functional activity of the labeled components should be tested and compared to the
unlabeled species; only those fluorescence derivatives should be used which have
properties that are sufficiently similar to the unmodified molecules.