Computational protein engineering: bridging the gap between rational design and laboratory evolution.

Enzymes are tremendously proficient catalysts, which can be used as extracellular catalysts for a whole host of processes, from chemical synthesis to the generation of novel biofuels. For them to be more amenable to the needs of biotechnology, however, it is often necessary to be able to manipulate their physico-chemical properties in an efficient and streamlined manner, and, ideally, to be able to train them to catalyze completely new reactions. Recent years have seen an explosion of interest in different approaches to achieve this, both in the laboratory, and in silico. There remains, however, a gap between current approaches to computational enzyme design, which have primarily focused on the early stages of the design process, and laboratory evolution, which is an extremely powerful tool for enzyme redesign, but will always be limited by the vastness of sequence space combined with the low frequency for desirable mutations. This review discusses different approaches towards computational enzyme design and demonstrates how combining newly developed screening approaches that can rapidly predict potential mutation "hotspots" with approaches that can quantitatively and reliably dissect the catalytic step can bridge the gap that currently exists between computational enzyme design and laboratory evolution studies.

Cellular polyamines promote amyloid-beta (Aß) peptide fibrillation and modulate the aggregation pathways.

The cellular polyamines spermine, spermidine, and their metabolic precursor putrescine, have long been associated with cell-growth, tumor-related gene regulations, and Alzheimer's disease. Here, we show by in vitro spectroscopy and AFM imaging, that these molecules promote aggregation of amyloid-beta (Aß) peptides into fibrils and modulate the aggregation pathways. NMR measurements showed that the three polyamines share a similar binding mode to monomeric Aß(1-40) peptide. Kinetic ThT studies showed that already very low polyamine concentrations promote amyloid formation: addition of 10 µM spermine (normal intracellular concentration is ~1 mM) significantly decreased the lag and transition times of the aggregation process. Spermidine and putrescine additions yielded similar but weaker effects. CD measurements demonstrated that the three polyamines induce different aggregation pathways, involving different forms of induced secondary structure. This is supported by AFM images showing that the three polyamines induce Aß(1-40) aggregates with different morphologies. The results reinforce the notion that designing suitable ligands which modulate the aggregation of Aß peptides toward minimally toxic pathways may be a possible therapeutic strategy for Alzheimer's disease.

Computational Study of the pKa Values of Potential Catalytic Residues in the Active Site of Monoamine Oxidase B.

Monoamine oxidase (MAO), which exists in two isozymic forms, MAO A and MAO B, is an important flavoenzyme responsible for the metabolism of amine neurotransmitters such as dopamine, serotonin, and norepinephrine. Despite extensive research effort, neither the catalytic nor the inhibition mechanisms of MAO have been completely understood. There has also been dispute with regard to the protonation state of the substrate upon entering the active site, as well as the identity of residues that are important for the initial deprotonation of irreversible acetylenic inhibitors, in accordance with the recently proposed mechanism. Therefore, in order to investigate features essential for the modes of action of MAO, we have calculated pKa values of three relevant tyrosine residues in the MAO B active site, with and without dopamine bound as the substrate (as well as the pKa of the dopamine itself in the active site). The calculated pKa values for Tyr188, Tyr398, and Tyr435 in the complex are found to be shifted upward to 13.0, 13.7, and 14.7, respectively, relative to 10.1 in aqueous solution, ruling out the likelihood that they are viable proton acceptors. The altered tyrosine pKa values could be rationalized as an interplay of two opposing effects: insertion of positively charged bulky dopamine that lowers tyrosine pKa values, and subsequent removal of water molecules from the active site that elevates tyrosine pKa values, in which the latter prevails. Additionally, the pKa value of the bound dopamine (8.8) is practically unchanged compared to the corresponding value in aqueous solution (8.9), as would be expected from a charged amine placed in a hydrophobic active site consisting of aromatic moieties. We also observed potentially favorable cation-π interactions between the -NH3(+) group on dopamine and aromatic moieties, which provide a stabilizing effect to the charged fragment. Thus, we offer here theoretical evidence that the amine is most likely to be present in the active site in its protonated form, which is similar to the conclusion from experimental studies of MAO A (Jones et al. J. Neural Trans.2007, 114, 707-712). However, the free energy cost of transferring the proton from the substrate to the bulk solvent is only 1.9 kcal mol(-1), leaving open the possibility that the amine enters the chemical step in its neutral form. In conjunction with additional experimental and computational work, the data presented here should lead toward a deeper understanding of mechanisms of the catalytic activity and irreversible inhibition of MAO B, which can allow for the design of novel and improved MAO B inhibitors.

Determination of the interaction between glimepiride and hyperbranched polymers in solid dispersions.

Solid dispersions of glimepiride, belonging to the sulfonylurea group of antidiabetic drugs, and poly(ester amide) hyperbranched polymers of different chemical compositions were prepared in order to improve glimepiride's poor water solubility. X-ray powder diffraction results show that glimepiride is in noncrystalline form, indicating that drug molecules are molecularly dispersed within the amorphous hyperbranched polymers. Nuclear magnetic resonance spectroscopy and Fourier transform-infrared spectroscopy results reveal the complex formation between the glimepiride drug and the particular hyperbranched polymer, which was confirmed also by quantum chemical calculations. The complex is stabilized by a hydrogen-bond interaction between the NH group of the sulfonylurea segment of glimepiride and the carbonyls of the amide and ester bonds of the hyperbranched polymers. The slightly acidic proton of the NH group of the sulfonylurea segment of glimepiride is also involved in an interaction with the tertiary amino functional groups of the hyperbranched polymer. As a consequence, the loading capacity is higher for the hyperbranched polymer with the tertiary amino groups. Owing to a complex formation between glimepiride and a particular hyperbranched polymer, glimepiride's water solubility and its dissolution rate are considerably improved relative to the pure glimepiride drug.