Destabilizing mutants

Fiqure 17.2 Denaturation of wild-type and two destabilized mutants of chymotrypsin inhibitor 2 (CI2) induced by gnanidinium chloride. Substituting equation 17.6 into equation 17.5 gives... 

McLean, A. J., Zeng, F. Y., Behan, D., Chalmers, D., and Milligan, G. (2002). Generation and analysis of constitutively active and physically destabilized mutants of the human beta(l)-adrenoceptor. Mol. Pharmacol. 62, 747-755. 

Backbone amide RDCs, the tryptophan Ne-H resonance and the amide nitrogen transverse relaxation rates for varying pH values and different temperatures have been measured by Ding et alf in their studies on equilibrium unfolding of a destabilized mutant of the B1 domain of protein G. The measured RDCs indicate that prior to complete unfolding, two melting hot spots are formed around Til, L12 and K13, and the N terminus of the helix at A24 and T25. 

Many proteins are capable of populating partially folded states under specific solution conditions and, occasionally, coexistence of the folded and an unfolded state can be observed by NMR. Ding et al have reported amide residual dipolar couplings (RDCs), nitrogen transverse relaxation rates for varying pH values and different temperatures on the destabilized mutant of the B1 domain of protein G (GBl). The RDCs for the low pH, thermally unfolded state of GBl are very small and do not indicate the presence of any native-like structure. Their data provided clear evidence for intermediate conformations and multi-state equilibrium unfolding for this GBl variant ... 

Fast-channel syndrome. The clinical features resemble those of autoimmune myasthenia gravis (see below) with variable severity. Conversely to what is found in slow-channel syndrome, the open state of the AChR is destabilized, manifesting as fast dissociation of ACh from the receptor and/or excessively reduced open times. One mutation has also caused multiple congenital joint contractures owing to fetal hypomotility in utero. In most cases, the mutant allele causing the kinetic abnormality... 

Sehgal According to some people, the per and timgal4 drivers are markers for all the neurons, or maybe even all the cells in the adult. I agree that they are not found everywhere, but the expression is widespread. One explanation is that the drivers are promiscuous. The other is that there are low levels of expression in these places that we don t detect any other way. I would think that this is kind of supported by some of the doubletime data, where, in a douhktime mutant we get per expression in ectopic locations. This would argue that per is synthesized in a lot of locations where you normally don t detect it because it is destabilized by doubletime. 

This hypothesis suggests that drug-resistant mutations would be of the type that destabilize protein structures. Such mutations are typically from larger to smaller side chains or from highly branched to less branched side chains [43-45]. Four of five observed compensation mutants are of this type (Table... 

Evolution by the selection of newly appearing mutants, which by virtue of a selective advantage disobey the condition crm > 1 and thus destabilize the dominance of the wild-type. 

Then z-scores for all mutant objects are calculated and the portion of stabilizing, destabilizing, and neutral mutations is recorded. These values are finally sent to the screen or to a file, if a filename was given as additional argument, analyse mutability Fd 9... 

The final session demonstrates how to characterize a protein region as random-izable. For a set of solvent-exposed residues of an immunoglobulin structure, 103 mutants are randomly generated. We examine how many of these mutants are destabilized with respect to the wild type. The analysis is repeated with an alternative set of residues that correspond to part of the natural epitope of the immunoglobulin structure. 

This command first derives 103 mutants from the object Fd by substituting all amino acids in the specified positions with randomly chosen ones. For each mutant, z-scores are calculated and compared to the Fd z-scores. Since 1001 z-scores have to be determined, this calculation lasts about 5.5 h. Results are written to two files Fd epil. sip contains all the z-scores, and Fd epil. nrm holds a summary of the number of stabilized, destabilized, and neutral mutants within the sample population (Figure 11.8). 

Here, the situation is almost reversed only a minority of the mutants (1.1%) exhibit a combined z-score that indicates destabilization (Figure 11.9 and 11.10 for a comparison of the z-score distributions). This result is in good agreement with our prior knowledge about immunoglobulin structures the amino acids of the epitope are supposed to be primarily selected for binding the antigen, rather than for their contribution to structural stability. 

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