Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas.

Carbonic anhydrase (CA) is one of nature's fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme's sensitivity to the harsh process conditions. Using directed evolution, the properties of a ß-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction.

Prostate tumor specific peptide-peptoid hybrid prodrugs.

Inspired by naturally occurring host defense peptides, cationic amphipathic peptoids provide a promising scaffold for anti-cancer therapeutics. Herein, we report a library of peptide-peptoid hybrid prodrugs that can be selectively activated by prostate cancer cells. We have identified several compounds demonstrating potent anti-cancer activity with good to moderate selectivity. We believe that these prodrugs can provide a useful design principle for next generation peptide-peptoid hybrid prodrugs.

Stability of biocatalysts.

Despite their many favorable qualities, the marginal stability of biocatalysts in many types of reaction media often has prevented or delayed their implementation for industrial-scale synthesis of fine chemicals and pharmaceuticals. Consequently, there is great interest in understanding effects of solution conditions on protein stability, as well as in developing strategies to improve protein stability in desired reaction media. Recent methods include novel chemical modifications of protein, lyophilization in the presence of additives, and physical immobilization on novel supports. Rational and combinatorial protein engineering techniques have been used to yield unmodified proteins with exceptionally improved stability. Both have been aided by the development of computational tools and structure-guided heuristics aimed at reducing library sizes that must be generated and screened to identify improved mutants. The number of parameters used to indicate protein stability can complicate discussions and investigations, and care should be taken to identify whether thermodynamic or kinetic stability limits the observed stability of proteins. Although the useful lifetime of a biocatalyst is dictated by its kinetic stability, only 6% of protein stability studies use kinetic stability measures. Clearly, more effort is needed to study how solution conditions impact protein kinetic stability.

Kinetic model for salt-induced protein deactivation.

A quantitative description of the influence of salts and buffer components on the degradation of proteins is important for the shelf life of pharmaceuticals and operating life of biocatalysts but is currently lacking. By modeling observed protein deactivation as the result of competing chaotrope-dependent and ion hydration-independent processes, we develop a model to fit experimental data and describe Hofmeister effects on the deactivation of a variety of proteins in chaotropic or kosmotropic aqueous solution. We demonstrate that four parameters are required to characterize loss of function of a protein in aqueous salt solution: (i) a protein-dependent kosmotropic deactivation constant k p, (ii) a chaotropic preexponential factor k c, (iii) an ion hydration coefficient omega, and (iv) the B-viscosity coefficient of the salt. This model fits our experimental data on horse-liver alcohol dehydrogenase (HL-ADH), alpha-chymotrypsin, and monomeric red fluorescent protein (mRFP). We calculate the kinetic m values ( m (double dagger)) to indicate whether the transition state of deactivation resembles the native state or the unfolded state. We find that the transition state of deactivation in a strongly chaotropic aqueous solution resembles the unfolded state (thermodynamic control) and infer that with decreasing chaotropicity the resemblance with the native state increases until at B approximately 0 kinetic control dominates. The developed model demonstrates the importance of ion hydration effects for the explanation of Hofmeister effects on proteins and leads to an expression for a kinetic equivalent of the Wyman linkage which can be used as an alternate method for calculating m (double dagger) parameters in aqueous solution at any salt composition and temperature with targeted experimental effort.

High-throughput screening for enhanced protein stability.

High thermostability of proteins is a prerequisite for their implementation in biocatalytic processes and in the evolution of new functions. Various protein engineering methods have been applied to the evolution of increased thermostability, including the use of combinatorial design where a diverse library of proteins is generated and screened for variants with increased stability. Current trends are toward the use of data-driven methods that reduce the library size by using available data to choose areas of the protein to target, without specifying the precise changes. For example, the half-lives of subtilisin and a Bacillus subtilis lipase were increased 1500-fold and 300-fold, respectively, using a crystal structure to guide mutagenesis choices. Sequence homology based methods have also produced libraries where 50% of the variants have improved thermostability. Moreover, advances in the high-throughput measurement of denaturation curves and the application of selection methods to thermostability evolution have enabled the screening of larger libraries. The combination of these methods will lead to the rapid improvement of protein stability for biotechnological purposes.

Evaluation of Hofmeister effects on the kinetic stability of proteins.

Dissolved salts are known to affect properties of proteins in solution including solubility and melting temperature, and the effects of dissolved salts can be ranked qualitatively by the Hofmeister series. We seek a quantitative model to predict the effects of salts in the Hofmeister series on the deactivation kinetics of enzymes. Such a model would allow for a better prediction of useful biocatalyst lifetimes or an improved estimation of protein-based pharmaceutical shelf life. Here we consider a number of salt properties that are proposed indicators of Hofmeister effects in the literature as a means for predicting salt effects on the deactivation of horse liver alcohol dehydrogenase (HL-ADH), alpha-chymotrypsin, and monomeric red fluorescent protein (mRFP). We find that surface tension increments are not accurate predictors of salt effects but find a common trend between observed deactivation constants and B-viscosity coefficients of the Jones-Dole equation, which are indicative of ion hydration. This trend suggests that deactivation constants (log k(d,obs)) vary linearly with chaotropic B-viscosity coefficients but are relatively unchanged in kosmotropic solutions. The invariance with kosmotropic B-viscosity coefficients suggests the existence of a minimum deactivation constant for proteins. Differential scanning calorimetry is used to measure protein melting temperatures and thermodynamic parameters, which are used to calculate the intrinsic irreversible deactivation constant. We find that either the protein unfolding rate or the rate of intrinsic irreversible deactivation can control the observed deactivation rates.

The Hofmeister effect on amyloid formation using yeast prion protein.

A variety of proteins are capable of converting from their soluble forms into highly ordered fibrous cross-beta aggregates (amyloids). This conversion is associated with certain pathological conditions in mammals, such as Alzheimer disease, and provides a basis for the infectious or hereditary protein isoforms (prions), causing neurodegenerative disorders in mammals and controlling heritable phenotypes in yeast. The N-proximal region of the yeast prion protein Sup35 (Sup35NM) is frequently used as a model system for amyloid conversion studies in vitro. Traditionally, amyloids are recognized by their ability to bind Congo Red dye specific to beta-sheet rich structures. However, methods for quantifying amyloid fibril formation thus far were based on measurements linking Congo Red absorbance to concentration of insulin fibrils and may not be directly applicable to other amyloid-forming proteins. Here, we present a corrected formula for measuring amyloid formation of Sup35NM by Congo Red assay. By utilizing this corrected procedure, we explore the effect of different sodium salts on the lag time and maximum rate of amyloid formation by Sup35NM. We find that increased kosmotropicity promotes amyloid polymerization in accordance with the Hofmeister series. In contrast, chaotropes inhibit polymerization, with the strength of inhibition correlating with the B-viscosity coefficient of the Jones-Dole equation, an increasingly accepted measure for the quantification of the Hofmeister series.