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Biomedical subjects

Frances H Arnold

Publications and source records attributed to Frances H Arnold.

At least 19 recordsLinked to original sources
PubMed2006

Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR.

The transcription factor LuxR activates gene expression in response to binding the signaling molecule 3-oxo-hexanoyl-homoserine lactone (3OC6HSL), an acyl-HSL with a carbonyl substituent at the third carbon of the acyl chain. We previously described a LuxR variant, LuxR-G2E, that activates gene expression by binding a broader range of acyl-HSLs, including straight-chain acyl-HSLs to which LuxR does not respond. Here, we use a dual positive-negative selection system to identify a variant of LuxR-G2E that retains the response to straight-chain acyl-HSLs, but no longer responds to 3OC6HSL. A single mutation, R67M, reduces LuxR-G2E's response to acyl-HSLs having a carbonyl substituent at the third carbon of the acyl chain. This specificity-enhancing mutation would not have been identified by positive selection alone. The dual selection system provides a rapid and reliable method for identifying LuxR variants that have or lack the desired response to a given set of acyl-HSL signals. LuxR variants with altered signaling specificities might become useful components for constructing artificial cell-cell communication systems that program population level behaviors.

Cell Communication↗
PubMed2006

Structure-guided recombination creates an artificial family of cytochromes P450.

Creating artificial protein families affords new opportunities to explore the determinants of structure and biological function free from many of the constraints of natural selection. We have created an artificial family comprising 3,000 P450 heme proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, is a valuable resource for sequence-structure-function studies. Logistic regression analysis of the multiple sequence alignment identifies key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2.

Amino Acid Sequence↗
PubMed2006

Protein stability promotes evolvability.

The biophysical properties that enable proteins to so readily evolve to perform diverse biochemical tasks are largely unknown. Here, we show that a protein's capacity to evolve is enhanced by the mutational robustness conferred by extra stability. We use simulations with model lattice proteins to demonstrate how extra stability increases evolvability by allowing a protein to accept a wider range of beneficial mutations while still folding to its native structure. We confirm this view experimentally by mutating marginally stable and thermostable variants of cytochrome P450 BM3. Mutants of the stabilized parent were more likely to exhibit new or improved functions. Only the stabilized P450 parent could tolerate the highly destabilizing mutations needed to confer novel activities such as hydroxylating the antiinflammatory drug naproxen. Our work establishes a crucial link between protein stability and evolution. We show that we can exploit this link to discover protein functions, and we suggest how natural evolution might do the same.

Base Pairing↗
PubMed2006

Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450.

Testing the toxicities and biological activities of the human metabolites of drugs is important for development of safe and effective pharmaceuticals. Producing these metabolites using human cytochrome P450s is difficult, however, because the human enzymes are costly, poorly stable, and slow. We have used directed evolution to generate variants of P450 BM3 from Bacillus megaterium that function via the "peroxide shunt" pathway, using hydrogen peroxide in place of the reductase domain, oxygen and NADPH. Here, we report further evolution of the P450 BM3 heme domain peroxygenase to enhance production of the authentic human metabolites of propranolol by this biocatalytic route. This system offers a versatile, cost-effective, and scaleable route to the synthesis of drug metabolites.

Bacillus megaterium↗
PubMed2006

Biosynthesis of novel carotenoid families based on unnatural carbon backbones: a model for diversification of natural product pathways.

We show that the C40 carotenoid desaturase CrtI from Pantoea ananatis (Erwinia uredovora) is capable of desaturating unnaturally long C45 and C50 carotenoid backbones in recombinant E. coli. Desaturation step number in these pathways is not very specific, and at least ten new C45 and C50 carotenoids were synthesized. We also present evidence for a novel asymmetric C40 backbone formed by the condensation of farnesyl diphosphate (C15PP) with farnesylgeranyl diphosphate (C25PP), and the subsequent desaturation of this backbone by CrtI in an atypical manner. Under some conditions, the C40, C45, and C50 carotenoid backbones synthesized in E. coli were monohydroxylated; their desaturation by CrtI in vitro led to yet more novel carotenoids. Challenging CrtI with larger-than-natural substrates in vivo has allowed us to show that this enzyme regulates desaturation step number by sensing the end groups of its substrate. Analysis of the mechanisms by which chemical diversity is generated and propagated through the nascent pathways provides insight into how natural product diversification occurs in nature.

Biological Products↗
PubMed2006

Enantioselective epoxidation of terminal alkenes to (R)- and (S)-epoxides by engineered cytochromes P450 BM-3.

Cytochrome P450 BM-3 from Bacillus megaterium was engineered for enantioselective epoxidation of simple terminal alkenes. Screening saturation mutagenesis libraries, in which mutations were introduced in the active site of an engineered P450, followed by recombination of beneficial mutations generated two P450 BM-3 variants that convert a range of terminal alkenes to either (R)- or (S)-epoxide (up to 83 % ee) with high catalytic turnovers (up to 1370) and high epoxidation selectivities (up to 95 %). A biocatalytic system using E. coli lysates containing P450 variants as the epoxidation catalysts and in vitro NADPH regeneration by the alcohol dehydrogenase from Thermoanaerobium brockii generates each of the epoxide enantiomers, without additional cofactor.

Journal Article↗
PubMed2005

Why highly expressed proteins evolve slowly.

Much recent work has explored molecular and population-genetic constraints on the rate of protein sequence evolution. The best predictor of evolutionary rate is expression level, for reasons that have remained unexplained. Here, we hypothesize that selection to reduce the burden of protein misfolding will favor protein sequences with increased robustness to translational missense errors. Pressure for translational robustness increases with expression level and constrains sequence evolution. Using several sequenced yeast genomes, global expression and protein abundance data, and sets of paralogs traceable to an ancient whole-genome duplication in yeast, we rule out several confounding effects and show that expression level explains roughly half the variation in Saccharomyces cerevisiae protein evolutionary rates. We examine causes for expression's dominant role and find that genome-wide tests favor the translational robustness explanation over existing hypotheses that invoke constraints on function or translational efficiency. Our results suggest that proteins evolve at rates largely unrelated to their functions and can explain why highly expressed proteins evolve slowly across the tree of life.

Computational Biology↗
PubMed2005

Why high-error-rate random mutagenesis libraries are enriched in functional and improved proteins.

The fraction of proteins that retain wild-type function after mutation has long been observed to decline exponentially as the average number of mutations per gene increases. Recently, several groups have used error-prone polymerase chain reactions (PCR) to generate libraries with 15 to 30 mutations per gene, on average, and have reported that orders of magnitude more proteins retain function than would be expected from the low-mutation-rate trend. Proteins with improved or novel function were isolated disproportionately from these high-error-rate libraries, leading to claims that high mutation rates unlock regions of sequence space that are enriched in positively coupled mutations. Here, we show experimentally that error-prone PCR produces a broader non-Poisson distribution of mutations consistent with a detailed model of PCR. As error rates increase, this distribution leads directly to the observed excesses in functional clones. We then show that while very low mutation rates result in many functional sequences, only a small number are unique. By contrast, very high mutation rates produce mostly unique sequences, but few retain function. Thus an optimal mutation rate exists that balances uniqueness and retention of function. Overall, high-error-rate mutagenesis libraries are enriched in improved sequences because they contain more unique, functional clones. Our findings demonstrate how optimal error-prone PCR mutation rates may be calculated, and indicate that "optimal" rates depend on both the protein and the mutagenesis protocol.

Data Interpretation, Statistical↗
PubMed2005

Long-term monitoring of bacteria undergoing programmed population control in a microchemostat.

Using an active approach to preventing biofilm formation, we implemented a microfluidic bioreactor that enables long-term culture and monitoring of extremely small populations of bacteria with single-cell resolution. We used this device to observe the dynamics of Escherichia coli carrying a synthetic "population control" circuit that regulates cell density through a feedback mechanism based on quorum sensing. The microfluidic bioreactor enabled long-term monitoring of unnatural behavior programmed by the synthetic circuit, which included sustained oscillations in cell density and associated morphological changes, over hundreds of hours.

4-Butyrolactone↗
PubMed2005

Protein-surfactant film voltammetry of wild-type and mutant cytochrome P450 BM3.

We are investigating the redox chemistry of wild-type (WT) and mutant (1-12G) cytochrome P450 BM3. Absorption spectra in solution feature the Fe(III) Soret at 418 nm for WT and a split Soret for 1-12G at 390 and 418 nm. Voltammetry of the proteins within DDAPSS films on the surface of carbon electrodes reveal nearly identical Fe(III/II) potentials (approximately -200 mV vs Ag/AgCl), but significant differences in k degrees , 250 vs 30 s(-)(1), and Fe(III/II)-CO potentials, -140 vs -115 mV, for WT vs 1-12G. Catalytic reduction of dioxygen by the proteins on rotating-disk electrodes was analyzed using Levich and Koutecky-Levich treatments. The data reveal 1-12G n and k(obs) values that are, respectively, 1.7 and 0.07 times those of WT, suggesting that the two proteins differ strikingly in their reactions with dioxygen.

Catalysis↗
PubMed2005

A synthetic multicellular system for programmed pattern formation.

Pattern formation is a hallmark of coordinated cell behaviour in both single and multicellular organisms. It typically involves cell-cell communication and intracellular signal processing. Here we show a synthetic multicellular system in which genetically engineered 'receiver' cells are programmed to form ring-like patterns of differentiation based on chemical gradients of an acyl-homoserine lactone (AHL) signal that is synthesized by 'sender' cells. In receiver cells, 'band-detect' gene networks respond to user-defined ranges of AHL concentrations. By fusing different fluorescent proteins as outputs of network variants, an initially undifferentiated 'lawn' of receivers is engineered to form a bullseye pattern around a sender colony. Other patterns, such as ellipses and clovers, are achieved by placing senders in different configurations. Experimental and theoretical analyses reveal which kinetic parameters most significantly affect ring development over time. Construction and study of such synthetic multicellular systems can improve our quantitative understanding of naturally occurring developmental processes and may foster applications in tissue engineering, biomaterial fabrication and biosensing.

4-Butyrolactone↗
PubMed2005

Stereoselective hydroxylation of an achiral cyclopentanecarboxylic acid derivative using engineered P450s BM-3.

Substrate engineered, achiral carboxylic acid derivative was biohydroxylated with various mutants of cytochrome P450 BM-3 to give two out of the four possible diastereoisomers in high de and ee. The BM-3 mutants exhibit up to 9200 total turnovers for hydroxylation of the engineered substrate, which without the protecting group is not transformed by this enzyme.

Bacterial Proteins↗
PubMed2005

On the conservative nature of intragenic recombination.

Intragenic recombination rapidly creates protein sequence diversity compared with random mutation, but little is known about the relative effects of recombination and mutation on protein function. Here, we compare recombination of the distantly related beta-lactamases PSE-4 and TEM-1 to mutation of PSE-4. We show that, among beta-lactamase variants containing the same number of amino acid substitutions, variants created by recombination retain function with a significantly higher probability than those generated by random mutagenesis. We present a simple model that accurately captures the differing effects of mutation and recombination in real and simulated proteins with only four parameters: (i) the amino acid sequence distance between parents, (ii) the number of substitutions, (iii) the average probability that random substitutions will preserve function, and (iv) the average probability that substitutions generated by recombination will preserve function. Our results expose a fundamental functional enrichment in regions of protein sequence space accessible by recombination and provide a framework for evaluating whether the relative rates of mutation and recombination observed in nature reflect the underlying imbalance in their effects on protein function.

Evolution, Molecular↗
PubMed2005

Thermodynamic prediction of protein neutrality.

We present a simple theory that uses thermodynamic parameters to predict the probability that a protein retains the wild-type structure after one or more random amino acid substitutions. Our theory predicts that for large numbers of substitutions the probability that a protein retains its structure will decline exponentially with the number of substitutions, with the severity of this decline determined by properties of the structure. Our theory also predicts that a protein can gain extra robustness to the first few substitutions by increasing its thermodynamic stability. We validate our theory with simulations on lattice protein models and by showing that it quantitatively predicts previously published experimental measurements on subtilisin and our own measurements on variants of TEM1 beta-lactamase. Our work unifies observations about the clustering of functional proteins in sequence space, and provides a basis for interpreting the response of proteins to substitutions in protein engineering applications.

Amino Acid Substitution↗
PubMed2005

Evolving strategies for enzyme engineering.

Directed evolution is a common technique to engineer enzymes for a diverse set of applications. Structural information and an understanding of how proteins respond to mutation and recombination are being used to develop improved directed evolution strategies by increasing the probability that mutant sequences have the desired properties. Strategies that target mutagenesis to particular regions of a protein or use recombination to introduce large sequence changes can complement full-gene random mutagenesis and pave the way to achieving ever more ambitious enzyme engineering goals.

Directed Molecular Evolution↗