Small Molecule-Protein Interaction enzyme

Since the conception of the two-hybrid assay to detect protein-protein interactions in vivo at the end of the 1980s, key modifications to this assay have expanded its scope to detect DNA-, RNA-, and small molecule-protein interactions in so-called n-hybrid assays. More recently, n-hybrid assays have also been used to detect enzyme catalysis, where enzyme activity is linked to cell survival via transcription of a reporter gene. Here we look at the initial publications that moved the two-hybrid assay into each of these new directions. 

In the present context a ligand (e.g. endogenous substances like hormones or neurotransmitters or xenobiotics like any synthetic drug) is defined as a small molecule which interacts with a macromolecular target like a protein receptor, a polynucleotide (DNA, RNA), or an enzyme... 

There are multiple sites of NO interaction with biochemical pathways, partly depending on the NO species encountered (Nathan, 1992 Stamler et al., 1992) (Table II). Nearly all cell components are possible targets, including proteins, carbohydrates, lipids, and nucleic acids, in addition to various small molecules. Most interactions of NO with enzymes cause inactivation, be it with heme groups, thiols, or other sites, with the notable exception of guanylate cyclase, which is stimulated by NO (Katsuki et al.. 

The analysis of myoglobin suggests that the native structure of a protein is often such that the small molecules that interact with the protein cannot enter or leave if the atoms are constrained to their average positions. Consequently, side chain and other fluctuations may be required for ligand binding by proteins and for the entrance of substrates and exit of products from enzymes. 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

Fig. 6.13. Different designs of FRET sensors. (A) Substrates for hydrolytic enzymes. (B) Sensors for bond formation. (C) Sensors based on conformational or structural change. (D) Environmentally sensitive probes. (E) Quenched activity-based probe to monitor small molecule-enzyme interaction. (F) Small molecule-enzyme interaction using a labeled protein.

The general types of protein-protein interactions that occur in cells include receptor-ligand, enzyme-substrate, multimeric complex formations, structural scaffolds, and chaperones. However, proteins interact with more targets than just other proteins. Protein interactions can include protein-protein or protein-peptide, protein-DNA/RNA or protein-nucleic acid, protein-glycan or protein-carbohydrate, protein-lipid or protein-membrane, and protein-small molecule or protein-ligand. It is likely that every molecule within a cell has some kind of specific interaction with a protein. 

Of the large number of protein interactions that take place in cells, perhaps the vast majority may be described as transient. Most proteins that modify other molecules do so very rapidly and so interact only briefly with their substrates or binding partners (i.e., enzymes). In addition, since proteins within cells are highly compartmentalized, the affinity of most interactions doesn t have to be very great, because each potential binding partner is within short diffusion distances and the relative concentration of molecules within these small volumes is high. 

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