Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design. C Elsevier Ltd. To carry out their activities, biological macromolecules balance different physical traits, such as stability, interaction affinity, and selectivity.
How such often opposing traits are encoded in a macromolecular system is critical to our understanding of evolutionary processes and ability to design new molecules with desired functions. We present a framework for constraining design simulations to balance different physical characteristics. Each trait is represented by the equilibrium fractional occupancy of the desired state relative to its alternatives, ranging from none to full occupancy, and the different traits are combined using Boolean operators to effect a "fuzzy"-logic language for encoding any combination of traits.
In another paper, we presented a new combinatorial backbone design algorithm AbDesign where the fuzzy-logic framework was used to optimize protein backbones and sequences for both stability and binding affinity in antibody-design simulation. We now extend this framework and find that fuzzy-logic design simulations reproduce sequence and structure design principles seen in nature to underlie exquisite specificity on the one hand and multispecificity on the other hand. The fuzzy-logic language is broadly applicable and could help define the space of tolerated and beneficial mutations in natural biomolecular systems and design artificial molecules that encode complex characteristics.
Published by Elsevier Ltd. The highly toxic organophosphorus OP nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. To investigate the suitability of the PTE mutant C23 as a catalytic scavenger, an in vivo guinea pig model was established to determine the efficacy of post-exposure treatment with C23 alone against VX intoxication.
Injection of C23 5 mg kg -1 i. A lower C23 dose 2 mg kg -1 reduced systemic toxicity and prevented mortality.
Delayed treatment i. Although performed under anesthesia, this proof-of-concept study demonstrated for the first time the ability of a catalytic bioscavenger to prevent systemic VX toxicity when given alone as a single postexposure treatment, and enables an initial assessment of a time window for this approach.
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In conclusion, the PTE mutant C23 may be considered as a promising starting point for the development of highly effective catalytic bioscavengers for post-exposure treatment of V-agents intoxication. All rights. We describe the expression and purification of DddD from the marine bacterium Marinomonas sp. MWYL1 and its biochemical characterization. These findings shed light on the biochemical utilization of DMSP in the marine environment. I discuss some physico-chemical and evolutionary aspects of enzyme accuracy selectivity, specificity end speed turnover rate, processivity.
Accuracy can be a beneficial side-product of active-sites being refined to proficiently convert a given substrate into one product. However, exclusion of undesirable, non-cognate substrates is also an explicitly evolved trait that may come with a cost. I define two schematic mechanisms. Ground-state discrimination applies to enzymes where selectivity is achieved primarily at the level of substrate binding.
Laboratory-Directed Protein Evolution
Exemplified by DNA methyltransferases and the ribosome, ground-state discrimination imposes strong accuracy-rate tradeoffs. Alternatively, transition-state discrimination, applies to relatively small substrates where substrate binding and chemistry are efficiently coupled, and evokes weaker tradeoffs.
Overall, the mechanistic, structural and evolutionary basis of enzymatic accuracy-rate tradeoffs merits deeper understanding. The potent human toxicity of organophosphorus OP nerve agents calls for the development of effective antidotes. Standard treatment for nerve agent poisoning with atropine and an oxime has a limited efficacy.
An alternative approach is the development of catalytic bioscavengers using OP-hydrolyzing enzymes such as paraoxonases PON1. Recently, a chimeric PON1 mutant, IIG1, was engineered toward the hydrolysis of the toxic isomers of soman and cyclosarin with high in vitro catalytic efficiency. In order to investigate the suitability of IIG1 as a catalytic bioscavenger, an in vivo guinea pig model was established to determine the protective effect of IIG1 against the highly toxic nerve agent cyclosarin.
Prophylactic i. A lower IIG1 dose 0. IIG1 exhibited a high catalytic efficiency with a homologous series of alkylmethylfluorophosphonates but had low efficiency with the phosphoramidate tabun and was virtually ineffective with the nerve agent VX. This quantitative analysis validated the model for predicting in vivo protection by catalytic bioscavengers based on their catalytic efficiency, the level of circulating enzyme, and the dose of the intoxicating nerve agent.
The in vitro and in vivo results indicate that IIG1 may be considered as a promising candidate bioscavenger to protect against the toxic effects of a range of highly toxic nerve agents. Are there structural features that make a fold amenable to functional innovation innovability? Do these features relate to robustness - the ability to readily accumulate sequence changes? We discuss several hypotheses regarding the relationship between the architecture of a protein and its evolutionary potential.
We hypothesize that polarity - differentiation and low connectivity between a protein's scaffold and its active-site - is a key prerequisite for innovability. Organismal adaptation to extreme temperatures yields enzymes with distinct configurational stabilities, including thermophilic and psychrophilic enzymes, which are adapted to high and low temperatures, respectively.
These enzymes are widely assumed to also have unique rate temperature dependencies.
Directed Evolution Library Creation: Methods and Protocols - Google книги
Thermophilic enzymes, for example, are considered optimal at high temperatures and effectively inactive at low temperatures due to excess rigidity. Surveying published data, we find that thermophilic, mesophilic, and psychrophilic enzymes exhibit indistinguishable rate temperature dependencies. Among other factors, this loss of rate acceleration may be ascribed to thermally induced vibrations compromising the activesite catalytic configuration, suggesting that many enzymes are in fact insufficiently rigid.
Protein engineering by directed evolution relies on the use of libraries enriched with beneficial variants. Such libraries should explore large mutational diversities while avoiding high loads of deleterious mutations. Here we describe a simple protocol for incorporating synthetic oligonucleotides that encode designed, site-specific mutations by assembly PCR.
This protocol enables a researcher to "hedge the bets," namely, to explore a large number of potentially beneficial mutations in a combinatorial manner such that individual library variants carry a limited number of mutations.
These organophosphates have a thiol leaving group with a choline-like moiety and are hydrolyzed very slowly by natural enzymes. We used an integrated computational and experimental approach to increase Brevundimonas diminuta phosphotriesterase's PTE detoxification rate of V-agents by fold. Computational models were built of the complex between PTE and V-agents. On the basis of these models, the active site was redesigned to be complementary in shape to VX and RVX and to include favorable electrostatic interactions with their choline-like leaving group.
Small libraries based on designed sequences were constructed. The libraries were screened by a direct assay for V-agent detoxification, as our initial studies showed that colorimetric surrogates fail to report the detoxification rates of the actual agents. The experimental results were fed back to improve the computational models. These new catalysts provide the basis for broad spectrum nerve agent detoxification.
Short insertions and deletions InDels comprise an important part of the natural mutational repertoire. InDels are, however, highly deleterious, primarily because two-thirds result in frame-shifts. However, the overall frequency of bypass and its relation to sequence composition remain unclear. Intriguingly, the occurrence of InDels and the bypass of frame-shifts are mechanistically related - occurring through slippage over repeats by DNA or RNA polymerases, or by the ribosome, respectively.
Here, we show that the frequency of frame-shifting InDels, and the frequency by which they are bypassed to give full-length, functional proteins, are indeed highly correlated. Using a laboratory genetic drift, we have exhaustively mapped all InDels that occurred within a single gene. We thus compared the naive InDel repertoire that results from DNA polymerase slippage to the frame-shifting InDels tolerated following selection to maintain protein function.
We found that InDels repeatedly occurred, and were bypassed, within homonucleotide repeats of bases. The longer the repeat, the higher was the frequency of InDels formation, and the more frequent was their bypass. Besides an expected 8A repeat, other types of repeats, including short ones, and G and C repeats, were bypassed.
Although obtained in vitro, our results indicate a direct link between the genetic occurrence of InDels and their phenotypic rescue, thus suggesting a potential role for frame-shifting InDels as bridging evolutionary intermediates. Serum paraoxonases PONs are detoxifying lactonases that were first identified in mammals. Three mammalian families are known, PON1, 2, and 3 that reside primarily in the liver. They catalyze essentially the same reaction, lactone hydrolysis, but differ in their substrate specificity.
Although some members are highly specific, others have a broad specificity profile. The evolutionary origins and substrate specificities of PONs therefore remain poorly understood. Here, we report a newly identified family of bacterial PONs, and the reconstruction of the ancestor of the three families of mammalian PONs. The mammalian PONs may therefore relate to a newly identified family of bacterial, PON-like "quorum-quenching" lactonases.
The appearance of PONs in metazoa is likely to relate to innate immunity rather than detoxification. Unlike the bacterial PON, the mammalian ancestor also hydrolyzes, with low efficiency, lactones other than homoserine lactones, thus preceding the detoxifying functions that diverged later in two of the three mammalian families. The bifunctionality of the mammalian ancestor and the trade-off between the quorum-quenching and detoxifying lactonase activities explain the broad and overlapping specificities of some mammalian PONs versus the singular specificity of others.
Alignments of orthologous protein sequences convey a complex picture. Some positions are utterly conserved whilst others have diverged to variable degrees. Amongst the latter, many are non-exchangeable between extant sequences. How do functionally critical and highly conserved residues diverge? Why and how did these exchanges become incompatible within contemporary sequences? Our model is phosphoglycerate kinase PGK , where lysine is an essential active-site residue completely conserved throughout Eukaryota and Bacteria, and serine is found only in archaeal PGKs.
Contemporary sequences tested exhibited complete loss of function upon exchanges at However, a directed evolution experiment revealed that two mutations were sufficient for human PGK to become functional with serine at position These two mutations made position permissive not only for serine and lysine, but also to a range of other amino acids seen in archaeal PGKs. The identified trajectories that enabled exchanges at show marked sign epistasis - a relatively small loss of function with respect to one amino acid lysine versus a large gain with another serine, and other amino acids.
Our findings support the view that, as theoretically described, the trajectories underlining the divergence of critical positions are dominated by sign epistatic interactions. Such trajectories are an outcome of rare mutational combinations. Nonetheless, as suggested by the laboratory enabled KS exchange, given enough time and variability in selection levels, even utterly conserved and functionally essential residues may change.
Protein evolvability includes two elements-robustness or neutrality, mutations having no effect and innovability mutations readily inducing new functions. How are these two conflicting demands bridged? Does the ability to bridge them relate to the observation that certain folds, such as TIM barrels, accommodate numerous functions, whereas other folds support only one?
Here, we hypothesize that the key to innovability is polarity-an active site composed of flexible, loosely packed loops alongside a well-separated, highly ordered scaffold. We show that highly stabilized variants of TEM-1 beta-lactamase exhibit selective rigidification of the enzyme's scaffold while the active-site loops maintained their conformational plasticity.