Protein binding, noncovalent

Proteins bind noncovalently to blot membranes. You can take the blotted protein off the membrane (e.g., for proteolytic digestion or for analysis in the MALDITOF). For nitrocellulose, Lui et al. (1996) have systematically examined the interaction of protein and blot membrane. According to them, Zwittergent 3-16 (1% in 100 mM NH4HCO3) removes between 60 and 90% of the blotted protein from the nitrocellulose. The detergent also works with PVDF membranes, albeit not as well. 

Figure 6 Effect of the increased rotational correlation time on the proton relaxivity of MP2269, a Gd111 chelate capable of noncovalent protein binding (Scheme 2). The lower NMRD curve was measured in water, whereas the upper curve was obtained in a 10%w/v bovine serum albumin solution in which the chelate is completely bound to the protein. The rotational correlation times calculated are rR=105ps in the nonbound state, and rR= 1,000 ps in the protein-bound state (t=35°C). For this chelate, the water exchange...

Antibodies against sugars (carbohydrate residues) can be difficult to obtain and lectins are a solution to these problems. Lectins are naturally occurring plant and animal proteins or glycoproteins that selectively bind noncovalently to carbohydrate residues. Lectins can be labeled directly or secondary antibodies against lectins enables the use of other immuno techniques (30) including electron microscopy (31). 

Data for the metabolites in plasma are generally for the unbound forms, but there is ample evidence that PPT bind noncovalently to proteins. Most studies on PPT-protein interaction have focused on protein utilization or astringency but a few studies have addressed binding to plasma proteins and lipoproteins. " " Strongest binding has been associated with 1,2-dihydroxyphenols and proline-rich proteins such as those character-istic of human saliva and structure-activity relationships have been reported. 

In protein microarrays, capture molecules need to be immobilized in a functional state on a solid support. In principle, the format of the assay system does not limit the choice of appropriate surface chemistry. The same immobilization procedure can be applied for both planar and bead-based systems. Proteins can be immobilized on various surfaces (Fig. 1) (12). Two-dimensional polystyrene, polylysine, aminosilane, or aldehyde, epoxy- or thiol group-coated surfaces can be used to immobilize proteins via noncovalent or covalent attachment (13,14). Three-dimensional supports like nitrocellulose or hydrogel-coated surfaces enable the immobilization of the proteins in a network structure. Larger quantities of proteins can be immobilized and kept in a functional state. Affinity binding reagents such as protein A, G, and L can be used to immobilize antibodies (15), streptavidin is used for biotinylated proteins (16), chelate for His-tagged proteins (17, 18), anti-GST antibodies for GST fusion proteins (19), and oligonucleotides for cDNA or mRNA-protein hybrids (20). 

Mechanism of activation of the epidermal growth factor (EGF) receptor, a representative receptor tyrosine kinase. The receptor polypeptide has extracellular and cytoplasmic domains, depicted above and below the plasma membrane. Upon binding of EGF (circle), the receptor converts from its inactive monomeric state (/eft) to an active dimeric state (right), in which two receptor polypeptides bind noncovalently. The cytoplasmic domains become phosphorylated (P) on specific tyrosine residues (Y) and their enzymatic activities are activated, catalyzing phosphorylation of substrate proteins (S). 

Definition of allosteric enzymes and their function Allosteric enzymes are multi-subunit proteins that are regulated by molecules called effectors. The effectors bind noncovalently at a site other than the active site, and can alter the affinity of the enzyme for its substrate, modify the maximal catalytic activity of the enzyme, or both. Allosteric enzymes frequently catalyze the committed step early in a pathway. 

One approach to the understanding of the relationship between the amino acid sequence of a protein and its three-dimensional structure consists of preparing fragments which reconstitute a functional nativelike structure by noncovalent association. Richards first demonstrated that the two fragments of bovine pancreatic ribonuelease, RNase-S-peptide (residues 1-20) and RNase-S-protein (residues 21-124), the latter with four intact disulfide bonds, bind noncovalently to form the original functional structure, RNase-S (73, 74)- The elucidation of the three-dimensional structure of RNase-S by X-ray crystallographic study confirmed these observations (75). The RNase-S-protein-RNase-S-peptide system also provided a way by which chemically synthesized fragments could be used to test the role of individual residues in the formation of the functional structure of the protein (76-79). 

Many biotransformations are simple functional group conversions, with rates dependent on a range of properties of the molecule and the organism. The various possible biotransformations, including spontaneous reactions, can be viewed as competing reactions kinetically slow biotransformations are frequently only apparent in the absence of alternative rapid biotransformations. Noncovalent protein binding of chemicals may reduce the availability for enzymic metabolism and... 

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