Proteins hydrophobic interactions

With hydrophobic interaction chromatography (HIC), hydrophobic proteins interact with slightly hydrophobic surfaces (propyl, ether, phenyl) at high salt concentrations. For elution, a salt gradient from high to low salt concentrations... 

The thioredoxin domain (see Figure 2.7) has a central (3 sheet surrounded by a helices. The active part of the molecule is a Pa(3 unit comprising p strands 2 and 3 joined by a helix 2. The redox-active disulfide bridge is at the amino end of this a helix and is formed by a Cys-X-X-Cys motif where X is any residue in DsbA, in thioredoxin, and in other members of this family of redox-active proteins. The a-helical domain of DsbA is positioned so that this disulfide bridge is at the center of a relatively extensive hydrophobic protein surface. Since disulfide bonds in proteins are usually buried in a hydrophobic environment, this hydrophobic surface in DsbA could provide an interaction area for exposed hydrophobic patches on partially folded protein substrates. 

Biomolecules interact with one another through molecular surfaces that are structurally complementary. How can various proteins interact with molecules as different as simple ions, hydrophobic lipids, polar but uncharged carbohydrates, and even nucleic acids ... 

Protein-protein interaction domain that recognizes short sequences containing a phosphotyrosine. Hydrophobic residues N-terminal to the phosphotyrosine residue provide distinction from SH2 domains. Particularly important in assembling protein complexes at activated receptors. 

Texturization is not measured directly but is inferred from the degree of denaturation or decrease of solubility of proteins. The quantities are determined by the difference in rates of moisture uptake between the native protein and the texturized protein (Kilara, 1984), or by a dyebinding assay (Bradford, 1976). Protein denaturation may be measured by determining changes in heat capacity, but it is more practical to measure the amount of insoluble fractions and differences in solubility after physical treatment (Kilara, 1984). The different rates of water absorption are presumed to relate to the degree of texturization as texturized proteins absorb water at different rates. The insolubility test for denaturation is therefore sometimes used as substitute for direct measurement of texturization. Protein solubility is affected by surface hydrophobicity, which is directly related to the extent of protein-protein interactions, an intrinsic property of the denatured state of the proteins (Damodaran, 1989 Vojdani, 1996). 

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... 

New developments in immobilization surfaces have lead to the use of SPR biosensors to monitor protein interactions with lipid surfaces and membrane-associated proteins. Commercially available (BIACORE) hydrophobic and lipophilic sensor surfaces have been designed to create stable membrane surfaces. It has been shown that the hydrophobic sensor surface can be used to form a lipid monolayer (Evans and MacKenzie, 1999). This monolayer surface can be used to monitor protein-lipid interactions. For example, a biosensor was used to examine binding of Src homology 2 domain to phosphoinositides within phospholipid bilayers (Surdo et al., 1999). In addition, a lipophilic sensor surface can be used to capture liposomes and form a lipid bilayer resembling a biological membrane. 

NHS-ester compounds to study protein interactions. These bis-NHS-PEG compounds may provide a superior crosslinker for studying such interactions due to their water solubility and the fact that the PEG bridge won t get buried in hydrophobic pockets on proteins or within hydrophobic membrane structures. 

Add a quantity of the Sulfo-SBED solution to the bait protein solution so that a 1- to 5-fold molar excess of crosslinker over the bait protein results in the reaction mixture. Mix well. Using greater quantities of Sulfo-SBED to the bait protein may result in precipitation due to the hydrophobic nature of crosslinker. In addition, over modification of the bait protein with the crosslinker may block sites of protein interaction, thus preventing complex formation. As a practical example, Horney et al. (2001), used a 1 1 molar ratio of Sulfo-SBED to the bait protein IGF-1 with success. 

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