Cold Adaptation of a Mesophilic Subtilisin-like Protease by Laboratory Evolution

Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts. In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart. A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (kcat) at 10 °C 6.6 times and a catalytic efficiency (kcat/KM) 9.6 times that of wild type. Its half-life at 70 °C is 3.3 times less than wild type. Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity. A first generation mutant with a >2-fold increase in kcat is actually more stable than wild type. This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability. SSII shares 77.4% identity with the naturally psychrophilic protease subtilisin S41. Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41. That none of the four are found in S41 indicates that there are multiple routes to cold adaptation

Directed evolution study of temperature adaptation in a psychrophilic enzyme

We have used laboratory evolution methods to enhance the thermostability and activity of the psychrophilic protease subtilisin S41, with the goal of investigating the mechanisms by which this enzyme can adapt to different selection pressures. A combined strategy of random mutagenesis, saturation mutagenesis and in vitro recombination (DNA shuf¯ing) was used to generate mutant libraries, which were screened to identify enzymes that acquired greater thermostability without sacri®cing lowtemperature activity. The half-life of seven-amino acid substitution variant 3-2G7 at 60 C is $500 times that of wild-type and far surpasses those of homologous mesophilic subtilisins. The dependence of half-life on calcium concentration indicates that enhanced calcium binding is largely responsible for the increased stability. The temperature optimum of the activity of 3-2G7 is shifted upward by$10 C. Unlike natural thermophilic enzymes, however, the activity of 3-2G7 at low temperatures was not compromised. The catalytic ef®ciency, k cat /K M , was enhanced $threefold over a wide temperature range (10 to 60 C). The activation energy for catalysis, determined by the temperature dependence of k cat /K M in the range 15 to 35 C, is nearly identical to wild-type and close to half that of its highly similar mesophilic homolog, subtilisin SSII, indicating that the evolved S41 enzyme retained its psychrophilic character in spite of its dramatically increased thermostability. These results demonstrate that it is possible to increase activity at low temperatures and stability at high temperatures simultaneously. The fact that enzymes displaying both properties are not found in nature most likely re¯ects the effects of evolution, rather than any intrinsic physicalchemical limitations on proteins

Common coding variant in SERPINA1 increases the risk for large artery stroke

Large artery atherosclerotic stroke (LAS) shows substantial heritability not explained by previous genome-wide association studies. Here, we explore the role of coding variation in LAS by analyzing variants on the HumanExome BeadChip in a total of 3,127 cases and 9,778 controls from Europe, Australia, and South Asia. We report on a nonsynonymous single-nucleotide variant in serpin family A member 1 (SERPINA1) encoding alpha-1 antitrypsin [AAT; p.V213A; P = 5.99E-9, odds ratio (OR) = 1.22] and confirm histone deacetylase 9 (HDAC9) as a major risk gene for LAS with an association in the 3?-UTR (rs2023938; P = 7.76E-7, OR = 1.28). Using quantitative microscale thermophoresis, we show that M1 (A213) exhibits an almost twofold lower dissociation constant with its primary target human neutrophil elastase (NE) in lipoprotein-containing plasma, but not in lipid-free plasma. Hydrogen/deuterium exchange combined with mass spectrometry further revealed a significant difference in the global flexibility of the two variants. The observed stronger interaction with lipoproteins in plasma and reduced global flexibility of the Val-213 variant most likely improve its local availability and reduce the extent of proteolytic inactivation by other proteases in atherosclerotic plaques. Our results indicate that the interplay between AAT, NE, and lipoprotein particles is modulated by the gate region around position 213 in AAT, far away from the unaltered reactive center loop (357-360). Collectively, our findings point to a functionally relevant balance between lipoproteins, proteases, and AAT in atherosclerosis

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances

Temperature adaptation of enzymes: Lessons from laboratory evolution

This chapter outlines the evolutionary protein design methods that are used to help uncover the molecular basis for temperature adaptation in enzymes. The chapter explains how temperature affects protein stability and enzyme activity. The chapter also discusses some of the results of comparative studies of enzymes isolated from the organisms adapted to different temperatures. The chapter reveals small number of studies on natural thermophilic proteins that has identified various thermodynamic strategies for stabilization. Laboratory evolution makes it possible to ask, for example, whether proteins have adopted these different strategies by chance, or whether certain protein architectures favor specific thermodynamic mechanisms. It will also be possible to determine how other selective pressures, such as the requirement for efficient low temperature activity, influence stabilization mechanisms. Directed evolution can also be used to probe the boundaries of protein function, for example, the role of protein stability in setting the upper temperature limits for life. The combination of directed evolution with high resolution structural studies and detailed characterization of dynamics promises to provide insights into the molecular basis of stability and catalysis

Temperature adaptation of enzymes: Lessons from laboratory evolution

This chapter outlines the evolutionary protein design methods that are used to help uncover the molecular basis for temperature adaptation in enzymes. The chapter explains how temperature affects protein stability and enzyme activity. The chapter also discusses some of the results of comparative studies of enzymes isolated from the organisms adapted to different temperatures. The chapter reveals small number of studies on natural thermophilic proteins that has identified various thermodynamic strategies for stabilization. Laboratory evolution makes it possible to ask, for example, whether proteins have adopted these different strategies by chance, or whether certain protein architectures favor specific thermodynamic mechanisms. It will also be possible to determine how other selective pressures, such as the requirement for efficient low temperature activity, influence stabilization mechanisms. Directed evolution can also be used to probe the boundaries of protein function, for example, the role of protein stability in setting the upper temperature limits for life. The combination of directed evolution with high resolution structural studies and detailed characterization of dynamics promises to provide insights into the molecular basis of stability and catalysis