Patrick L. Wintrode

How enzymes adapt: lessons from directed evolution

Enzymes that are adapted to widely different temperature niches are being used to investigate the molecular basis of protein stability and enzyme function. However, natural evolution is complex: random noise, historical accidents and ignorance of the selection pressures at work during adaptation all cloud comparative studies. Here, we review how adaptation in the laboratory by directed evolution can complement studies of natural enzymes in the effort to understand stability and function. Laboratory evolution experiments can attempt to mimic natural evolution and identify different adaptive mechanisms. However, laboratory evolution might make its biggest contribution in explorations of nonnatural functions, by allowing us to distinguish the properties nutured by evolution from those dictated by the laws of physical chemistry.

Temperature adaptation of enzymes: Lessons from laboratory evolution

Publisher Summary 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.

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 (k cat) at 10 °C 6.6 times and a catalytic efficiency (k cat/K M ) 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 k cat 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.

Protein Dynamics in a Family of Laboratory Evolved Thermophilic Enzymes

Molecular dynamics simulations were employed to study how protein solution structure and dynamics are affected by adaptation to high temperature. Simulations were carried out on a para-nitrobenzyl esterase (484 residues) and two thermostable variants that were generated by laboratory evolution. Although these variants display much higher melting temperatures than wild-type (up to 18 degrees C higher) they are both >97% identical in sequence to the wild-type. In simulations at 300 K the thermostable variants remain closer to their crystal structures than wild-type. However, they also display increased fluctuations about their time-averaged structures. Additionally, both variants show a small but significant increase in radius of gyration relative to wild-type. The vibrational density of states was calculated for each of the esterases. While the density of states profiles are similar overall, both thermostable mutants show increased populations of the very lowest frequency modes (<10 cm(-1)), with the more stable mutant showing the larger increase. This indicates that the thermally stable variants experience increased concerted motions relative to wild-type. Taken together, these data suggest that adaptation for high temperature stability has resulted in a restriction of large deviations from the native state and a corresponding increase in smaller scale fluctuations about the native state. These fluctuations contribute to entropy and hence to the stability of the native state. The largest changes in localized dynamics occur in surface loops, while other regions, particularly the active site residues, remain essentially unchanged. Several mutations, most notably L313F and H322Y in variant 8G8, are in the region showing the largest increase in fluctuations, suggesting that these mutations confer more flexibility to the loops. As a validation of our simulations, the fluctuations of Trp102 were examined in detail, and compared with Trp102 phosphorescence lifetimes that were previously measured. Consistent with expectations from the theory of phosphorescence, an inverse correlation between out-of-plane fluctuations on the picosecond time scale and phosphorescence lifetime was observed.

Folding mechanism of the metastable serpin α1-antitrypsin

The misfolding of serpins is linked to several genetic disorders including emphysema, thrombosis, and dementia. During folding, inhibitory serpins are kinetically trapped in a metastable state in which a stretch of residues near the C terminus of the molecule are exposed to solvent as a flexible loop (the reactive center loop). When they inhibit target proteases, serpins transition to a stable state in which the reactive center loop forms part of a six-stranded β-sheet. Here, we use hydrogen-deuterium exchange mass spectrometry to monitor region-specific folding of the canonical serpin human α1-antitrypsin (α1-AT). We find large differences in the folding kinetics of different regions. A key region in the metastable → stable transition, β-strand 5A, shows a lag phase of nearly 350 s. In contrast, the “B-C barrel” region shows no lag phase and the incorporation of the C-terminal residues into β-sheets B and C is largely complete before the center of β-sheet A begins to fold. We propose this as the mechanism for trapping α1-AT in a metastable form. Additionally, this separation of timescales in the folding of different regions suggests a mechanism by which α1-AT avoids polymerization during folding.

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type α1-antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the α1-antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of β-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into β-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of β-sheet C. On the basis of our results, we propose a mechanism for polymerization in which β-strand 1C is displaced from the rest of β-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of β-sheet A, and allows for subsequent polymerization.

Local and Global Effects of a Cavity Filling Mutation in a Metastable Serpin

The serpins are an unusual class of protease inhibitors which fold to a metastable form and subsequently undergo a massive conformational change to a stable form when they inhibit their target proteases. The driving force for this conformational change has been extensively investigated by site directed mutagenesis, and it has been found that mutations which stabilize the metastable form frequently result in activity deficiency. Here, we employ hydrogen/deuterium exchange to probe the effects of a cavity filling mutant of alpha(1)AT. The Gly117 --> Phe substitution fills a cavity between the F-helix and the face of beta-sheet A, stabilizes the metastable form of alpha(1)AT by approximately 4 kcal/mol and results in a 60% reduction in inhibitory activity against elastase. Globally, the G117F substitution alters the unfolding mechanism by eliminating the molten globule intermediate that is seen in wild type unfolding. Remarkably, this is accomplished primarily by destabilizing the molten globule rather than stabilizing the metastable native state. Locally, conformational flexibility in the native state is reduced in specific regions: the top of the F-helix, beta-strands 5A, 1C, and 4C, and helix D. Except for strand 4C, all of these regions mediate or propagate conformational changes. The F-helix and strand 5A must be displaced during protease inhibition, displacement of strand 1C is required for polymer formation, and helix D is a site (in antithrombin) of allosteric regulation. Our results indicate that these functionally important regions form a delocalized network of residues that are dynamically coupled and that both local and global stability mediate inhibitory activity.

Native State Dynamics of Human Neuroserpin Investigated with Hydrogen/Deuterium Exchange and Molecular Dynamics Simulations

Wild-type human neuroserpin, a member of the serine protease inhibitor superfamily, is expressed in neurons of the central and peripheral nervous system, as well as in the adult brain. Polymerization of certain mutants of neuroserpin is associated with dementia caused by familial encephalopathy. We have performed hydrogen/deuterium exchange-mass spectrometry in order to monitor the structural stability and flexibility of different regions of the neuroserpin structure. We find that beta-sheet A, a critical region thought to be involved in polymerization, is less stable and more labile in neuroserpin than in other serpins such as alpha-1 antitrypsin and antithrombin. This may explain why wild-type neuroserpin is more susceptible to polymerization than other serpins. Molecular dynamics simulations also indicate that Wild Type neuroserpin shows increases flexibility on the nanosecond timescale as compared with alpha-1 antitrypsin. In the simulations, a novel 2 stranded beta-sheet was formed between the N terminal portion of the reactive center loop and the loop connecting strand 3A to beta-sheet C. This phenomenon occurred repeatedly in multiple independent simulations. If such an interaction in fact occurs in solution, it could contribute to the relatively poor inhibitory efficiency of neuroserpin compared to other serpins by retarding the insertion of the reactive center loop into sheet A after proteolytic cleavage. Simulations of a pathological mutant of neuroserpin showed distortions near the top of the central beta-sheet A, a critical site for polymer formation. This distortion may help explain why the mutant is more prone to polymerize than wild type.