CiteULike: Author Hellinga

Protein fabrication automation.

JC Cox — 2007-10-31T16:19:39-00:00

Protein Sci, Vol. 16, No. 3. (March 2007), pp. 379-390.

Facile "writing" of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de novo construction of any desired ORF from oligonucleotides with low effort, high speed, and little human interaction. PFA comprises software for sequence design, data management, and the generation of instruction sets for liquid-handling robotics, a liquid-handling robot, a robust PCR scheme for gene assembly from synthetic oligonucleotides, and a genetic selection system to enrich correctly assembled full-length synthetic ORFs. The process is robust and scalable.

Computational design of a biologically active enzyme.

MA Dwyer — 2005-11-09T14:17:29-00:00

Science, Vol. 304, No. 5679. (25 June 2004), pp. 1967-1971.

Rational design of enzymes is a stringent test of our understanding of protein chemistry and has numerous potential applications. Here, we present and experimentally validate the computational design of enzyme activity in proteins of known structure. We have predicted mutations that introduce triose phosphate isomerase activity into ribose-binding protein, a receptor that normally lacks enzyme activity. The resulting designs contain 18 to 22 mutations, exhibit 10(5)- to 10(6)-fold rate enhancements over the uncatalyzed reaction, and are biologically active, in that they support the growth of Escherichia coli under gluconeogenic conditions. The inherent generality of the design method suggests that many enzymes can be designed by this approach.

Generalized dead-end elimination algorithms make large-scale protein side-chain structure prediction tractable: implications for protein design and structural genomics.

LL Looger — 2005-10-04T20:47:21-00:00

J Mol Biol, Vol. 307, No. 1. (16 March 2001), pp. 429-445.

The dead-end elimination (DEE) theorems are powerful tools for the combinatorial optimization of protein side-chain placement in protein design and homology modeling. In order to reach their full potential, the theorems must be extended to handle very hard problems. We present a suite of new algorithms within the DEE paradigm that significantly extend its range of convergence and reduce run time. As a demonstration, we show that a total protein design problem of 10(115) combinations, a hydrophobic core design problem of 10(244) combinations, and a side-chain placement problem of 10(1044) combinations are solved in less than two weeks, a day and a half, and an hour of CPU time, respectively. This extends the range of the method by approximately 53, 144 and 851 log-units, respectively, using modest computational resources. Small to average-sized protein domains can now be designed automatically, and side-chain placement calculations can be solved for nearly all sizes of proteins and protein complexes in the growing field of structural genomics.

Variable Speed Limits: Safety and Operational Impacts of a Candidate Control Strategy for Freeway Applications

P Allaby — 2008-05-19T10:19:43-00:00

Intelligent Transportation Systems, IEEE Transactions on, Vol. 8, No. 4. (2007), pp. 671-680.

Variable-speed limit sign (VSLS) systems enable transportation managers to dynamically change the posted speed limit in response to prevailing traffic and/or weather conditions. Although VSLSs have been implemented in a limited number of jurisdictions throughout the world, there is currently very limited documentation that describes quantitative safety and operational impacts. Furthermore, the impacts reported are primarily from systems in Europe and may not be directly transferable to other jurisdictions such as North America. This paper presents the results of an evaluation of a candidate VSLS system for an urban freeway in Toronto, ON, Canada. The evaluation was conducted using a microscopic simulation model combined with a categorical crash potential model for estimating safety impacts.

Converting a maltose receptor into a nascent binuclear copper oxygenase by computational design.

DE Benson — 2008-04-27T14:26:10-00:00

Biochemistry, Vol. 41, No. 9. (5 March 2002), pp. 3262-3269.

Computational protein design methods were used to identify mutations that are predicted to introduce a binuclear copper center coordinated by six histidines, replacing the maltose-binding site in Escherichia coli maltose-binding protein (MBP) with an oxygen-binding site. A small family of five candidate designs consisting of 9 to 10 mutations each was constructed by oligonucleotide-directed mutagenesis. These mutant proteins were expressed and purified, and their stability, copper- and cobalt-binding properties, and interactions of the resulting metalloprotein complexes with azide, hydrogen peroxide, and dioxygen were characterized. We identified one 10-fold mutant, MBP.Hc.E, that can form Cu(II)(2) and Co(II)(2) complexes that interact with H(2)O(2) and O(2). The Co(II)(2) protein reacts with H(2)O(2) to form a complex that is spectroscopically similar to a synthetic model that structurally mimics the oxy-hemocyanin core, whereas the Cu(II)(2) protein reacted with O(2) or H(2)O(2) does not. We postulate that the equilibrium between the open and closed conformations of MBP allows species with variable Cu-Cu distances to form, and that such species can bind ligands in geometries that are not observed in natural type III centers. Introduction of one additional mutation in the hinge region of MBP, I329F, known to favor formation of the closed state, results in a binuclear copper center that when reacted with low concentrations of H(2)O(2) mimics the spectroscopic signature of oxy-hemocyanin.

Rational design of nascent metalloenzymes.

DE Benson — 2008-04-27T14:25:38-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 97, No. 12. (6 June 2000), pp. 6292-6297.

Understanding the early genesis of new enzymatic functions is one of the challenges in protein design, mechanistic enzymology, and molecular evolution. We have experimentally mimicked starting points in this process by introducing primitive iron and oxygen binding sites at various locations in thioredoxin, a small protein lacking metal centers, by using computational design. These rudimentary active sites show emerging enzymatic activities that select to varying degrees between different oxygen chemistries. Even within these nascent enzymes, mechanisms by which different reactions are controlled can be discerned. These involve both stabilizing and destabilizing interactions imposed on the metal center by the surrounding protein matrix.

The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors.

JS Marvin — 2008-04-25T09:19:27-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 94, No. 9. (29 April 1997), pp. 4366-4371.

Rational protein design is an emerging approach for testing general theories of structure and function. The ability to manipulate function rationally also offers the possibility of creating new proteins of biotechnological value. Here we use the design approach to test the current understanding of the structural principles of allosteric interactions in proteins and demonstrate how a simple allosteric system can form the basis for the construction of a generic biosensor molecular engineering system. We have identified regions in Escherichia coli maltose-binding protein that are predicted to be allosterically linked to its maltose-binding site. Environmentally sensitive fluorophores were covalently attached to unique thiols introduced by cysteine mutations at specific sites within these regions. The fluorescence of such conjugates changes cooperatively with respect to maltose binding, as predicted. Spatial separation of the binding site and reporter groups allows the intrinsic properties of each to be manipulated independently. Provided allosteric linkage is maintained, ligand binding can therefore be altered without affecting transduction of the binding event by fluorescence. To demonstrate applicability to biosensor technology, we have introduced a series of point mutations in the maltose-binding site that lower the affinity of the protein for its ligand. These mutant proteins have been combined in a composite biosensor capable of measuring substrate concentration within 5% accuracy over a concentration range spanning five orders of magnitude.

Optimal Sequence Selection in Proteins of Known Structure by Simulated Evolution

HW Hellinga — 2008-04-22T11:48:36-00:00

Proceedings of the National Academy of Sciences, Vol. 91, No. 13. (21 June 1994), pp. 5803-5807.

10.1073/pnas.91.13.5803

The backbone structure of the thermophilic Thermoanaerobacter tengcongensis ribose binding protein is essentially identical to its mesophilic E. coli homolog

Matthew Cuneo — 2008-03-28T18:03:01-00:00

BMC Structural Biology, Vol. 8 (28 March 2008), 20.

Construction of new ligand binding sites in proteins of known structure. I. Computer-aided modeling of sites with pre-defined geometry.

HW Hellinga — 2007-10-11T19:25:57-00:00

J Mol Biol, Vol. 222, No. 3. (5 December 1991), pp. 763-785.

We have devised a molecular model building computer program (DEZYMER) which builds new ligand binding sites into a protein of known three-dimensional structure. It alters only the sequence and the side-chain structure of the protein, leaving the protein backbone fold intact by definition. The program searches for a constellation of backbone positions arranged such that if appropriate side-chains were placed there, they would bind the ligand according to a pre-defined geometry of interaction specified by the experimentalist. These binding sites are introduced by the program by taking into account simple rules such as steric hindrance, atomic close-packing and hydrogen bond patterns, which are known to maintain the integrity of a protein structure to a first approximation. A test case is presented in this paper where the copper binding site found in blue-copper proteins such as plastocyanin, azurin and cupredoxin is introduced into Escherichia coli thioredoxin. The model building of one of the solutions found by the program is presented in some detail. The experimental construction and properties of this new protein are described in an accompanying paper. It is hoped that this program provides a general method for the design of ligand binding sites and enzyme active sites, which can then be tested experimentally.

The construction of metal centers in proteins by rational design.

HW Hellinga — 2007-10-11T18:54:28-00:00

Fold Des, Vol. 3, No. 1. (1998)

Metalloprotein properties result from the interplay between coordination requirements of the metal center, protein stability, and modulation of the metal center by the surrounding protein matrix. Simple metal centers, which exercise control over the protein by affecting stability or enzyme activity, have been created by rational design. Complex centers, which require control by the protein matrix, have also been constructed.

Strategy for dynamic process modeling based on neural networks in macroscopic balances

Henricus Van Can — 2007-03-24T23:32:17-00:00

AIChE Journal, Vol. 42, No. 12. (1996), pp. 3403-3418.

Gray box models combine the short development time of data-driven black box models with extrapolation properties of knowledge-driven first principles models (white box), which in (bio)chemical engineering are always based on macroscopic balances. By modeling the inaccurately known terms in a macroscopic balance with a black box model, one naturally obtains a so-called serial gray box model configuration. The identification data must cover only the input-output space of the inaccurately known terms, and the accurately known terms can be used for reliable extrapolation. In this way, the serial gray box configuration results in accurate models with known extrapolation properties with a limited experimental effort. This strategy is demonstrated for the modeling and control of a pressure vessel using real-time experiments. For this case, the strategy is superior to a black box modeling approach that requires much more data and to a parallel gray box approach that results in a model with poor extrapolation properties. Moreover, neural networks are an accurate and convenient modeling tool for the black part in gray box model configurations, because a very fast noniterative training algorithm is used for training neural networks.

An efficient model development strategy for bioprocesses based on neural networks in macroscopic balances

HJL van Can — 2007-03-24T23:31:12-00:00

Biotechnology and Bioengineering, Vol. 54, No. 6. (1997), pp. 549-566.

In the serial gray box modeling strategy, generally available knowledge, represented in the macroscopic balance, is combined naturally with neural networks, which are powerful and convenient tools to model the inaccurately known terms in the macroscopic balance. This article shows, for a typical biochemical conversion, that in the serial gray box modeling strategy the identification data only have to cover the input-output space of the inaccurately known term in the macroscopic balances and that the accurately known terms can be used to achieve reliable extrapolation. The strategy is demonstrated successfully on the modeling of the enzymatic (repeated) batch conversion of penicillin G, for which real-time results are presented. Compared with a more data-driven black box strategy, the serial gray box strategy leads to models with reliable extrapolation properties, so that with the same number of identification experiments the model can be applied to a much wider range of different conditions. Compared to a more knowledge-driven white box strategy, the serial gray box model structure is only based on readily available or easily obtainable knowledge, so that the development time of serial gray box models still may be short in a situation where there is no detailed knowledge of the system available. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 549-566, 1997.

Computational design of receptor and sensor proteins with novel functions.

LL Looger — 2006-04-04T16:43:19-00:00

Nature, Vol. 423, No. 6936. (8 May 2003), pp. 185-190.

The formation of complexes between proteins and ligands is fundamental to biological processes at the molecular level. Manipulation of molecular recognition between ligands and proteins is therefore important for basic biological studies and has many biotechnological applications, including the construction of enzymes, biosensors, genetic circuits, signal transduction pathways and chiral separations. The systematic manipulation of binding sites remains a major challenge. Computational design offers enormous generality for engineering protein structure and function. Here we present a structure-based computational method that can drastically redesign protein ligand-binding specificities. This method was used to construct soluble receptors that bind trinitrotoluene, l-lactate or serotonin with high selectivity and affinity. These engineered receptors can function as biosensors for their new ligands; we also incorporated them into synthetic bacterial signal transduction pathways, regulating gene expression in response to extracellular trinitrotoluene or l-lactate. The use of various ligands and proteins shows that a high degree of control over biomolecular recognition has been established computationally. The biological and biosensing activities of the designed receptors illustrate potential applications of computational design.

Periplasmic binding proteins: a versatile superfamily for protein engineering

Mary Dwyer — 2006-01-02T14:32:48-00:00

Current Opinion in Structural Biology, Vol. 14, No. 4. (August 2004), pp. 495-504.

The diversity of biological function, ligand binding, conformational changes and structural adaptability of the periplasmic binding protein superfamily have been exploited to engineer biosensors, allosteric control elements, biologically active receptors and enzymes using a combination of techniques, including computational design. Extensively redesigned periplasmic binding proteins have been re-introduced into bacteria to function in synthetic signal transduction pathways that respond to extracellular ligands and as biologically active enzymes.

Control of a biomolecular motor-powered nanodevice with an engineered chemical switch.

H Liu — 2005-11-03T03:56:08-00:00

Nat Mater, Vol. 1, No. 3. (November 2002), pp. 173-177.

The biophysical and biochemical properties of motor proteins have been well-studied, but these motors also show promise as mechanical components in hybrid nano-engineered systems. The cytoplasmic F(1) fragment of the adenosine triphosphate synthase (F1-ATPase) can function as an ATP-fuelled rotary motor and has been integrated into self-assembled nanomechanical systems as a mechanical actuator. Here we present the rational design, construction and analysis of a mutant F1-ATPase motor containing a metal-binding site that functions as a zinc-dependent, reversible on/off switch. Repeated cycles of zinc addition and removal by chelation result in inhibition and restoration, respectively, of both ATP hydrolysis and motor rotation of the mutant, but not of the wild-type F1 fragment. These results demonstrate the ability to engineer chemical regulation into a biomolecular motor and represent a critical step towards controlling integrated nanomechanical devices at the single-molecule level.

Conversion of a maltose receptor into a zinc biosensor by computational design.

JS Marvin — 2005-03-11T16:06:39-00:00

Proc Natl Acad Sci U S A, Vol. 98, No. 9. (24 April 2001), pp. 4955-4960.

We have demonstrated that it is possible to radically change the specificity of maltose binding protein by converting it into a zinc sensor using a rational design approach. In this new molecular sensor, zinc binding is transduced into a readily detected fluorescence signal by use of an engineered conformational coupling mechanism linking ligand binding to reporter group response. An iterative progressive design strategy led to the construction of variants with increased zinc affinity by combining binding sites, optimizing the primary coordination sphere, and exploiting conformational equilibria. Intermediates in the design series show that the adaptive process involves both introduction and optimization of new functions and removal of adverse vestigial interactions. The latter demonstrates the importance of the rational design approach in uncovering cryptic phenomena in protein function, which cannot be revealed by the study of naturally evolved systems.