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Efficient design of functional proteins with higher thermal stability remains challenging especially for highly diverse sequence variants. Considering the evolutionary pressure on protein folds, sequence design optimizing evolutionary fitness could help designing folds with higher stability. Using a generative evolution fitness model trained to capture variation patterns in natural sequences, we designed artificial sequences of a proteinaceous inhibitor of pectin methylesterase enzymes. These inhibitors have considerable industrial interest to avoid phase separation in fruit juice manufacturing or reduce methanol in distillates, averting chromatographic passages triggering unwanted aroma loss. Six out of seven designs with up to 30 % divergence to other inhibitor sequences are functional and two have improved thermal stability. This method can improve protein stability expanding functional protein sequence space, with traits valuable for industrial applications and scientific research. [ABSTRACT FROM AUTHOR]

HB43 (FAKLLAKLAKKLL) is a synthetic peptide active against cell lines derived from breast, colon, melanoma, lung, prostate, and cervical cancers. Despite its remarkable spectrum of activity, the mechanism of action at the molecular level has never been investigated, preventing further optimization of its selectivity. The alternation of charged and hydrophobic residues suggests amphipathicity, but the formation of alpha‐helical structure seems discouraged by its short length and the large number of positively charged residues. Using different biophysical and in silico approaches we show that HB43 is completely unstructured in solution but assumes alpha‐helical conformation in the presence of DPC micelles and liposomes exposing phosphatidylserine (PS) used as mimics of cancer cell membranes. Membrane permeabilization assays demonstrate that the interaction leads to the preferential destabilization of PS‐containing vesicles with respect to PC‐containing ones, here used as noncancerous cell mimics. ssNMR reveals that HB43 is able to fluidify the internal structure of cancer‐cell mimicking liposomes while MD simulations show its internalization in such bilayers. This is achieved by the formation of specific interactions between the lysine side chains and the carboxylate group of phosphatidylserine and/or the phosphate oxygen atoms of targeted phospholipids, which could catalyze the formation of the alpha helix required for internalization. With the aim of better understanding the peptide biocompatibility and the additional antibacterial activity, the interaction with noncancerous cell mimicking liposomes exposing phosphatidylcholine (PC) and bacterial mimicking bilayers exposing phosphatidylglycerol (PG) is also described. [ABSTRACT FROM AUTHOR]