Site protein stability

Hl-loop cation-binding sites can stabilize the flexible [14] a 1 helix designated as the thumb epitope, which is spatially close to the wrist epitope (Fig. 6d). This segment is found only among the TGF-b isoforms and the activins but not in other members of the TGF-b superfamily and may also participate in the recognition of type I receptor or other TGF-b activity regulation proteins, such as betaglycan. 

The ability to biosynthetically incorporate noncoded amino acids into proteins site-specifically has facilitated studies not previously possible. These include studies of protein stability, the initiation of protein translation, electron transfer, protein-protein and protein-membrane interactions, reversal of enzyme substrate specificity, and structure-function relationships, among others. A growing number of research labs have begun to report applications of this technique. A brief look at some recent applications of the suppression mutagenesis technique follows. 

Replication starts by the separation of the strands of DNA and the formation of a local bubble at a specific DNA site called the origin of replication (ori). A helicase enzyme uses energy from ATP hydrolysis to effect this action. Single-strand DNA binding proteins stabilize the strands during the subsequent steps. The original DNA strands will function as the templates that will direct synthesis of the complementary strands. A nucleotide on the template strand will determine which deoxyribonucleotide (dNTP) will be incorporated in the newly synthesized strand. This replica-... 

Since there are strict stereochemical requirements for the relative positions and orientations of the two participating cysteine residues,11 addition of new disulfides to existing proteins by site-directed mutagenesis has not always produced the desired increase in stability. Introduction of disulfide bonds has been attempted for phage T4 lysozyme,4-71 phage A repressor,81 dihydrofolate reductase,91 and subtilisins.10-131 Among them the most extensive study has been performed on T4 lysozyme, and enhancement of protein stability has been successful. 

At present, 16 cysteine-containing subtilisin-type enzymes are known and the position of the cysteine residues is restricted to the nine corresponding sites described above.42 Of the 16 enzymes, six enzymes other than aqualysin I and proteinase K have cysteine residues at positions where the cysteine residues are able to form disulfide bond(s) like the two enzymes. Although these disulfide bonds seem to have been acquired to increase protein stability, only four kinds of disulfide bonds are found in the subtilisin-type enzymes, suggesting that the positions of the disulfide bonds have been selected strictly in the process of molecular evolution of the enzyme. 

Because of the strict stereochemical requirements, it is not easy to find optimal sites for the introduction of disulfide bonds into proteins. Introduction of disulfide bonds into T4 lysozyme has been engineered by theoretical calculations and computer modeling.4 7 The results obtained from the mutant lysozymes illustrate several points relevant to the use of disulfide bonds for improving protein stability.6 (i) Introduction of the cysteine(s) should minimize the disruption or loss of interactions that stabilize the native structure, (ii) The size of the loop formed by the crosslink should be as large as possible, (iii) The strain energy introduced by the disulfide bond should be kept as low as possible. For this purpose, a location within the flexible part of the molecule is desirable. 

If certain amino acids (such as disulfide bonds or crucial amino acids in the hydrophobic core) are indispensable for protein stability, these positions can be changed by site-directed mutagenesis (Proba et al., 1998). To avoid back-mutations during the evolution process or the selection of a residual wild-type contamination, the pool is amplified after each round of ribosome display with a primer that reintroduces the destabilizing mutation. If the mutation is not close to one of the termini, the coding sequence has to be amplified in two parts, which are then reassembled by PCR. Thus, to evolve improved stabilities this strategy first removes known crucial stabilizing factors to select for compensatory mutations at different positions. 

The theory could successfully explain the restabilization of protein-stabilized polymer latexes the increase in surface dipole density with increasing electrolyte concentration, generated by the binding of the counterions to sites of opposite charge, is in this case responsible for this effect This occurs because the repulsive force generated by the surface dipoles more than compensates for the decrease in repulsion caused by screening. 

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