Thermally stable active engineered proteins can be used as a starting point in directed evolution experiments to evolve new functions on the background of this additional “reservoir of stability.” The stabilized enzyme may be able to accumulate a greater number of destabilizing yet functionally important mutations before unfolding, protease digestion, and aggregation abolish its activity.

mutations had a destabilizing effect according to all three metrics.

These mutants, along with the destabilized outlier 1155A for which relative Tm depends on simulation length (Fig.

The likely explanation of the distinction between an apparent tradeoff when mutations are made in the active site and the opposite trend for mutations outside of the active site is that “carving” an active site requires special selection of catalytic amino acids, which could indeed have a destabilizing effect, overall.

Our earlier study [48] analyzed a smaller set of primarily destabilizing mutants and did not reveal any statistically significant trend (positive or negative) in the stability-activity relation for DHFR.

In 4/ 16 of the experimentally stabilizing mutations, a residue was changed to the consensus residue, while only 2/29 destabilizing mutations resulted from a change to consensus.

Likewise, in 18/29 destabilizing mutations, a residue was changed away from the consensus residue, while this was true for only 5/ 16 of stabilizing mutations.

While most mutations in a natural protein are destabilizing [8,9] , biological proteins are not generally at their highest possible stability; some mutations will stabilize a protein, increasing the equilibrium population of the folded state [10—12].

Apparently, protein loci where mutations can cause significant stabilization are statistically less susceptible to destabilizing mutations and vice versa, which may be expected: once a residue is already at its most stabilizing amino acid variant, the protein cannot be stabilized further by mutation.

Distinct outliers correspond to the loci with the strongest stabilizing or destabilizing effects of mutations.

If we consider only the binary prediction of Whether a mutation is stabilizing or destabilizing , MCPU can correctly predict 11 out of 16 mutations, While Eris and SDM correctly classify 9 and 8 mutations respectively.

Notably, all the connected ligands within the subnetwork shared a similar microtubule destabilization effect.

Identification of a compound subnetwork with a consensus tubulin destabilizing effect, related to Fig.

(A) Mapping of tubulin polymerization activity onto the mitotic compound set CSN identified a compound subnetwork with a consensus tubulin destabilization effect.

(B) Tubulin polymerization kinetics for 7 novel tubulin destabilizers (6—12), based on a phenyl-sulfanyl-thiazol-acetamide scaffold, using an in-vitro tubulin polymerization assay.

To test CSNAP’s prediction that 51 of the 212 mitotic compounds were targeting microtubules, we reacquired all 212 compounds and tested their ability to perturb micro-tubule polymerization (stabilize or destabilize microtubules) in an in-vitro microtubule polymerization assay at 50uM concentration (Fig.

In contrast, the lipopeptides readily insert into the inner membrane core, and the concomitant increased hydration may be responsible for bilayer destabilization and antimicrobial function.

Nevertheless, it has been widely documented that divalent cations are essential for maintenance of OM stability, and PMB1 has been shown to cause destabilisation via displacement of these divalent cations [6, 31, 32].

In this case, deep penetration of monomeric PMBl molecules enables the DAB residues to drag water into the membrane, suggesting an alternative antimicrobial mechanism for IM destabilisation .