Novel 1:1 Labeling and Purification Process for C‑Terminal Thioester and Single Cysteine Recombinant Proteins Using Generic Peptidic Toolbox Reagents
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Abstract
We
developed a versatile set of chemical labeling reagents which
allow dye ligation to the C-terminus of a protein or a single internal
cysteine and target purification in a simple two-step process. This
simple process results in a fully 1:1 labeled conjugate suitable for
all quantitative fluorescence spectroscopy and imaging experiments.
We refer to a “generic labeling toolbox” because of
the flexibility to choose one of many available dyes, spacers of different
lengths and compositions which increase the target solubility, a variety
of affinity purification tags, and different cleavage chemistries
to release the 1:1 labeled proteins. Studying protein function in
vitro or in the context of live cells and organisms is of vital importance
in biological research. Although label free detection technologies
gain increasing interest in molecular recognition science, fluorescence
spectroscopy is still the most often used detection technique for
assays and screens both in academic as well as in industrial groups.
For generations, fluorescence spectroscopists have labeled their proteins
of interest with small fluorescent dyes by random chemical linking
on the proteins’ exposed lysines and cysteines. Chemical reactions
with a certain excess of activated esters or maleimides of longer
wavelength dyes hardly ever result in quantitative labeling of the
target protein. Most of the time, more than one exposed amino acid
side chain reacts. This results in a mixture of dye–protein
complexes of different labeling stoichiometries and labeling sites.
Only mass spectrometry allows resolving the precise chemical composition
of the conjugates. In “classical” ensemble averaging
fluorescent experiments, these labeled proteins are still useful,
and quantification of, e.g., ligand binding experiments, is achieved
via knowledge of the overall protein concentration and a fluorescent
signal change which is proportional to the amount of complex formed.
With the development of fluorescence fluctuation analysis techniques
working at single molecule resolution, like fluorescence correlation
spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS),
fluorescence intensity diffusion analysis (FIDA), etc., it became
important to work with homogeneously labeled target proteins. Each
molecule participating in a binding equilibrium should be detectable
when it freely fluctuates through the confocal focus of a microscope.
The measured photon burst for each transition contains information
about the size and the stoichiometry of a protein complex. Therefore,
it is important to work with reagents that contain an exact number
of tracers per protein at identical positions. The ideal fluorescent
tracer–protein complex stoichiometry is 1:1. While genetic
tags such as fluorescent proteins (FPs) are widely used to detect
proteins, FPs have several limitations compared to chemical tags.
For example, FPs cannot easily compete with organic dyes in the flexibility
of modification and spectral range; moreover, FPs have disadvantages
in brightness and photostability and are therefore not ideal for
most biochemical single molecule studies. We present the synthesis
of a series of exemplaric toolbox reagents and labeling results on
three target proteins which were needed for high throughput screening
experiments using fluorescence fluctuation analysis at single molecule
resolution. On one target, Hu-antigen R (HuR), we demonstrated the
activity of the 1:1 labeled protein in ribonucleic acid (RNA) binding,
and the ease of resolving the stoichiometry of an RNA-HuR complex
using the same dye on protein and RNA by Fluorescence Intensity Multiple
Distribution Analysis (FIMDA) detection