Protein complexes, nonspecific

Lerche, M.H. and Poulsen, F.M. (1998) Solution structure of barley lipid transfer protein complexed with palmitate. Two different binding modes of palmitate in the homologous maize and barley nonspecific lipid transfer proteins. Protein Science 7, 2490-2498. 

If naked DNA (that is, DNA that is not protein-complexed) is partially digested with a nonspecific endonuclease that randomly cuts double strands, a broad smear of polynucleotide fragments is observed in an electrophoresis gel. If the same experiment is conducted with chromatin, or even with whole nuclei (which the nuclease can easily penetrate through the nuclear pores), the random DNA cleavage yields a series of bands that are multiples of approximately 200 base pairs. This indicates that "naked" DNA is present only at regularly spaced points. 

Certain proteins, such as insulin, transferrin, and insulin-like growth factors, cross the blood-brain barrier by receptor-mediated transcytosis. Once the protein binds to its membrane receptor, the membrane containing the receptor-protein complex is endocytosed into the endothelial cell to form a vesicle. It is released on the other side of the endothelial cell. Absorptive-mediated transcytosis also can occur. It differs from receptor-mediated transcytosis in that the protein binds nonspecifically to the membrane and not to a distinct receptor. 

It is well known that there are electrostatic interactions between redox proteins in the physiological electron transfer (ET) chains. The initial step in the formation of a protein complex is a nonspecific association between two proteins, followed by a rotational diffusion on the molecular surface to reach a proper configuration for the ET reaction. Details of the molecular structure of such complexes and intermolecular ET reactions have not been fully understood by using physiological redox complexes because of the complexity of biological systems. 

Fig. 1 Schematics of tandem affinity purification (TAP). The methodology of using TAP technique for protein complex purification is outlined in 6 steps. (1) The target protein (the bait ) is co-transcribed with the tandem-arranged affinity tags to either the N- or the C-terminus of the gene (shown is the N-terminal fusion). The two affinity tags (shaded boxes 1 and 2j are separated by a protease cleavage site (blank bo)(j, from where the far-end tag would be cut off and the near-end one would be exposed to the affinity columns for a second-round purification. (2) The bait and the tags are expressed as a chimeric fusion protein. The bait would form a complex with its in vivo partners (depicted as A, B, and Q. Nonspecific association may happen as well, as depicted by D, E, and F. (3) The first round purification via specific interaction between the affinity binder 1 and the far-end tag eliminates most nonspecific associated proteins. (4) In the second round purification, the near-end tag is cut off and the protein complex is further purified, with most nonspecific associates removed. (5 and 6) The bait protein and its associated proteins are eluted from the column and are further analyzed either by using antibodies, or by mass spectrometry...

Similar results were obtained with a different but less sensitive test system (76,102), utilizing 0.5 pg of I-labeled F(ab )2 fingments of IgG myeloma protein and rabbit antiidiotypic antiserum. Complexes were precipitated by goat anti-rabbit Fc. The ratio of the inhibitory capacity of homologous protein to nonspecific IgG exceeded 3 x 1(1 to I (102). 

Footprinting With respect to molecular genetics, a technique used to identify the DNA segment in contact with a given DNA-binding protein. The DNA-protein complex is subjected to digestion with a nonspecific nuclease, which cleaves at the residues that are not protected by the protein. 

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