A fluorogenic derivatization method was developed to distinguish the protein N-terminal acetylation status. The unacetylated protein selectively reacted with 4-chloro-7-nitrobenzofurazan (NBD-Cl) at neutral pH to provide high fluorescence. In contrast, the protein with N-terminal acetylation was essentially nonfluorescent under the same conditions despite the presence of many internal lysine residues. Fluorescence of the NBD-labeled protein was very stable, and only micromolar concentrations of proteins were required for reliable detection. This method also provides a general and practical way to quantify proteins when their N-terminal amino group is available.
"Another popular fluorescent probe is 7-nitrobenz-2- oxa-1,3-diazole (NBD). Since its introduction in 1968 , this polaritysensitive probe has been increasingly used for labeling lipids   , proteins            , oligonucleotides , and drugs  , because its chloride and fluoride derivatives react easily with thiol and amino groups leading to stable fluorescent adducts. NBD derivatives have long been utilized in studies of artificial and biological membranes. "
[Show abstract][Hide abstract] ABSTRACT: The environmentally sensitive fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole (NBD) is generally utilized to monitor dynamic properties of membrane lipids and proteins. Here we studied the behavior of a homologous series of 4-n-alkylamino-substituted NBD derivatives (NBD-Cn; n=4, 6, 8, 9, 10, 12) in planar lipid bilayers, liposomes and isolated mitochondria. NBD-C10 induced proton conductivity in planar lipid membranes, while NBD-C4 was ineffective. The NBD-Cn compounds readily provoked proton permeability of neutral liposomes being less effective in negatively charged liposomes. NBD-Cn increased the respiration rate and reduced the membrane potential of isolated rat liver mitochondria. Remarkably, the bell-shaped dependence of the uncoupling activity of NBD-Cn on the alkyl chain length was found in mitochondria in contrast to the monotonous dependence in liposomes. The effect of NBD-Cn on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by mitochondria swelling. Binding of NBD-Cn to mitochondria increased with n, as shown by fluorescence correlation spectroscopy. It was concluded that despite a pKa value of the amino group in NBD-Cn being about 10, i.e. far from the physiological pH range, the expected hindering of the uncoupling activity could be overcome by inserting the alkyl chain of a certain length.
[Show abstract][Hide abstract] 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 "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 results 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 homogenously 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 are 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.
[Show abstract][Hide abstract] ABSTRACT: Fluorescent dyes are used to investigate the hydrophobic exposure of proteins in different conformational states. Despite of their wider applications, the molecular level interactions of these dyes with different states of proteins are not well understood. In this report, we have analyzed the interaction between proteins and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) using molecular docking and simulation. Three different unfolded conformations of alpha-lactalbumin (alpha LA) and ribonuclease A (RNase A) were generated. TNS was docked against these protein states and the molecular simulation of protein-TNS complexes were carried out. These results were compared with TNS in water and TNS bound to the native form of bovine serum albumin (BSA). The results suggest that TNS binds to the proteins through ionic and hydrophobic interactions. TNS bound to the unfolded conformations of alpha LA and RNase A and the native-BSA has less solvent accessibility and surrounded by more hydrophobic residues. The orientation of TNS in protein bound states were analyzed using two of its bond angles and dihedrals. The analysis shows that the aromatic rings of TNS move from near planar to non-planar in different protein-TNS complexes and ring rotation is much constrained. Orientation of TNS is influenced by the conformation of the binding pocket and the hydrogen bonding interactions between the protein and TNS. Therefore, we suggest that TNS fluorescence enhancement upon binding to unfolded states of proteins might be due to the hydrophobic environment and reduced solvent accessibility rather than the binding ability and specific orientation of TNS in the bound state.
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