The de novo design of protein receptors and catalysts requires the development of new and functional polypeptides. As one approach, minimized protein receptors may be abstracted from larger, natural contexts. To explore the utility of employing computational methods for this approach, we experimentally evaluated the previously reported design of a minimal NAD+-binding domain based on the structure of Squalus acanthias lactate dehydrogenase. Synthesis of an appropriate gene and characterization of the resulting protein revealed little secondary structure and no discernible tertiary structure. Evaluation and redesign of the protein using inverse folding methods resulted in only modest improvements. We have attempted to direct the in vitro evolution of artificial antibodies based on alternative protein scaffolds. Diverse libraries of potential receptors were prepared by randomizing two loops in the nucleotide binding site of flavodoxin from Desulfovibrio vulgaris. Selection for novel binding function was then pursued using phage display technology, which mimics the immune system in vitro. The inability to obtain functional receptors by this approach is discussed and future directions are proposed. As an alternative to building new function into existing protein domains, we have also exploited pre-existing function to analyze the determinants of protein structure and to redesign protein architecture. Genetic selection provides a potentially general means of obtaining active catalysts from a diverse population of variants. We show how this tool can be employed for the rapid and sensitive evaluation of protein structure. Through combinatorial mutagenesis and selection, we explored sequence constraints on an interhelical turn in the enzyme chorismate mutase from Escherichia coli. We were able to uncover subtle amino acid preferences not observed in previous studies. In addition, we found that long-range tertiary interactions between residues in a turn and residues elsewhere in the protein can impose a severe constraint on the number of acceptable amino acids at these locations. We exploited genetic selection for the purposes of protein design by transforming the intimately entwined, dimeric chorismate mutase from Methanococcus jannaschii into a monomeric, four-helix-bundle protein with high catalytic activity. The success of this endeavor can be attributed to the choice of a thermostable starting protein, the introduction of point mutations that preferentially destabilize the dimeric structure, and the use of directed evolution to optimize the sequence of an inserted interhelical turn. We found that less than 0.05% of turn sequences yielded well-behaved, monomeric, and highly active proteins. (Abstract shortened by UMI.)

This is the first high-quality, comprehensive overview of the field of evolutionary protein design. Topics include new protein design strategies, the structures of laboratory-evolved proteins, the evolution of non-natural enzyme functions, and the theory of laboratory evolution.

The Arthur M. Sackler Colloquia of the National Academy of Sciences address scientific topics of broad and current interest, cutting across the boundaries of traditional disciplines. Each year, four or five such colloquia are scheduled, typically two days in length and international in scope. Colloquia are organized by a member of the Academy, often with the assistance of an organizing committee, and feature presentations by leading scientists in the field and discussions with a hundred or more researchers with an interest in the topic. Colloquia presentations are recorded and posted on the National Academy of Sciences Sackler colloquia website and published on CD-ROM. These Colloquia are made possible by a generous gift from Mrs. Jill Sackler, in memory of her husband, Arthur M. Sackler.

Directed evolution comprises two distinct steps that are typically applied in an iterative fashion: (1) generating molecular diversity and (2) finding among the ensemble of mutant sequences those proteins that perform the desired fu- tion according to the specified criteria. In many ways, the second step is the most challenging. No matter how cleverly designed or diverse the starting library, without an effective screening strategy the ability to isolate useful clones is severely diminished. The best screens are (1) high throughput, to increase the likelihood that useful clones will be found; (2) sufficiently sen- tive (i. e. , good signal to noise) to allow the isolation of lower activity clones early in evolution; (3) sufficiently reproducible to allow one to find small improvements; (4) robust, which means that the signal afforded by active clones is not dependent on difficult-to-control environmental variables; and, most importantly, (5) sensitive to the desired function. Regarding this last point, almost anyone who has attempted a directed evolution experiment has learned firsthand the truth of the dictum “you get what you screen for. ” The protocols in Directed Enzyme Evolution describe a series of detailed p- cedures of proven utility for directed evolution purposes. The volume begins with several selection strategies for enzyme evolution and continues with assay methods that can be used to screen enzyme libraries. Genetic selections offer the advantage that functional proteins can be isolated from very large libraries s- ply by growing a population of cells under selective conditions.

A one-stop reference that reviews protein design strategies to applications in industrial and medical biotechnology Protein Engineering: Tools and Applications is a comprehensive resource that offers a systematic and comprehensive review of the most recent advances in the field, and contains detailed information on the methodologies and strategies behind these approaches. The authors—noted experts on the topic—explore the distinctive advantages and disadvantages of the presented methodologies and strategies in a targeted and focused manner that allows for the adaptation and implementation of the strategies for new applications. The book contains information on the directed evolution, rational design, and semi-rational design of proteins and offers a review of the most recent applications in industrial and medical biotechnology. This important book: Covers technologies and methodologies used in protein engineering Includes the strategies behind the approaches, designed to help with the adaptation and implementation of these strategies for new applications Offers a comprehensive and thorough treatment of protein engineering from primary strategies to applications in industrial and medical biotechnology Presents cutting edge advances in the continuously evolving field of protein engineering Written for students and professionals of bioengineering, biotechnology, biochemistry, Protein Engineering: Tools and Applications offers an essential resource to the design strategies in protein engineering and reviews recent applications.

The aim this volume is to present the methods, challenges, software, and applications of this widespread and yet still evolving and maturing field. Computational Protein Design, the first book with this title, guides readers through computational protein design approaches, software and tailored solutions to specific case-study targets. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Computational Protein Design aims to ensure successful results in the further study of this vital field.

This book focuses on some of the most significant advances in enzyme engineering that have been achieved through directed evolution and hybrid approaches. On the 25th anniversary of the discovery of directed evolution, this volume is a tribute to the pioneers of this thrilling research field, and at the same time provides a comprehensive overview of current research and the state of the art. Directed molecular evolution has become the most reliable and robust method to tailor enzymes, metabolic pathways or even whole microorganisms with improved traits. By mirroring the Darwinian algorithm of natural selection on a laboratory scale, new biomolecules of invaluable biotechnological interest can now be engineered in a manner that surpasses the boundaries of nature. The volume is divided into two sections, the first of which provides an update on recent successful cases of enzyme ensembles from different areas of the biotechnological spectrum, including tryptophan synthases, unspecific peroxygenases, phytases, therapeutic enzymes, stereoselective enzymes and CO2-fixing enzymes. This section also provides information on the directed evolution of whole cells. The second section of the book summarizes a variety of the most applicable methods for library creation, together with the future trends aimed at bringing together directed evolution and in silico/computational enzyme design and ancestral resurrection.

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