Homme Wytzes Hellinga

Journal Article J Mol Biol · November 15, 2024 ABC transporters are ancient and ubiquitous nutrient transport systems in bacteria and play a central role in defining lifestyles. Periplasmic solute-binding proteins (SBPs) are components that deliver ligands to their translocation machinery. SBPs have di ... Full text Link to item Cite

Chromophore carbonyl twisting in fluorescent biosensors encodes direct readout of protein conformations with multicolor switching.

Journal Article Commun Chem · August 19, 2023 Fluorescent labeling of proteins is a powerful tool for probing structure-function relationships with many biosensing applications. Structure-based rules for systematically designing fluorescent biosensors require understanding ligand-mediated fluorescent ... Full text Link to item Cite

Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics.

Journal Article Nucleic Acids Res · June 23, 2023 DNA polymerases are essential for nucleic acid synthesis, cloning, sequencing and molecular diagnostics technologies. Conditional intein splicing is a powerful tool for controlling enzyme reactions. We have engineered a thermal switch into thermostable DNA ... Full text Link to item Cite

Structure-Guided Discovery of Potent Antifungals that Prevent Ras Signaling by Inhibiting Protein Farnesyltransferase.

Journal Article J Med Chem · October 27, 2022 Infections by fungal pathogens are difficult to treat due to a paucity of antifungals and emerging resistances. Next-generation antifungals therefore are needed urgently. We have developed compounds that prevent farnesylation of Cryptoccoccus neoformans Ra ... Full text Link to item Cite

Discovery of Thermostable, Fluorescently Responsive Glucose Biosensors by Structure-Assisted Function Extrapolation.

Journal Article Biochemistry · February 15, 2022 Accurate assignment of protein function from sequence remains a fascinating and difficult challenge. The periplasmic-binding protein (PBP) superfamily present an interesting case of function prediction because they are both ubiquitous in prokaryotes and te ... Full text Link to item Cite

Harnessing Environmental Ca2+ for Extracellular Protein Thermostabilization.

Journal Article Biochemistry · October 6, 2020 Ca2+ is the third-most prevalent metal ion in the environment. EF hands are common Ca2+-binding motifs found in both extracellular and intracellular proteins of eukaryotes and prokaryotes. Cytoplasmic EF hand proteins often mediate allosteric control of si ... Full text Link to item Cite

Describing Complex Structure-Function Relationships in Biomolecules at Equilibrium.

Journal Article J Mol Biol · March 27, 2020 One of the great ambitions of structural biology is to describe structure-function relationships quantitatively. Statistical thermodynamics is a powerful, general tool for computing the behavior of biological macromolecules at equilibrium because it establ ... Full text Link to item Cite

Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I.

Journal Article Proc Natl Acad Sci U S A · June 6, 2017 Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication p ... Full text Link to item Cite

Crystal structures of the fungal pathogen Aspergillus fumigatus protein farnesyltransferase complexed with substrates and inhibitors reveal features for antifungal drug design.

Journal Article Protein Sci · March 2014 Species of the fungal genus Aspergillus are significant human and agricultural pathogens that are often refractory to existing antifungal treatments. Protein farnesyltransferase (FTase), a critical enzyme in eukaryotes, is an attractive potential target fo ... Full text Link to item Cite

Visualization of synaptic inhibition with an optogenetic sensor developed by cell-free protein engineering automation.

Journal Article J Neurosci · October 9, 2013 We describe an engineered fluorescent optogenetic sensor, SuperClomeleon, that robustly detects inhibitory synaptic activity in single, cultured mouse neurons by reporting intracellular chloride changes produced by exogenous GABA or inhibitory synaptic act ... Full text Link to item Cite

Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides.

Journal Article J Biol Chem · August 17, 2012 In addition to discriminating against base pair mismatches, DNA polymerases exhibit a high degree of selectivity for deoxyribonucleotides over ribo- or dideoxynucleotides. It has been proposed that a single active site residue (steric gate) blocks producti ... Full text Link to item Cite

Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis.

Journal Article Proc Natl Acad Sci U S A · October 25, 2011 Even though high-fidelity polymerases copy DNA with remarkable accuracy, some base-pair mismatches are incorporated at low frequency, leading to spontaneous mutagenesis. Using high-resolution X-ray crystallographic analysis of a DNA polymerase that catalyz ... Full text Link to item Cite

Structures of Cryptococcus neoformans protein farnesyltransferase reveal strategies for developing inhibitors that target fungal pathogens.

Journal Article J Biol Chem · October 7, 2011 Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. ... Full text Link to item Cite

Quantitation of protein-protein interactions by thermal stability shift analysis.

Journal Article Protein Sci · August 2011 Thermal stability shift analysis is a powerful method for examining binding interactions in proteins. We demonstrate that under certain circumstances, protein-protein interactions can be quantitated by monitoring shifts in thermal stability using thermodyn ... Full text Link to item Cite

Integration of cell-free protein coexpression with an enzyme-linked immunosorbent assay enables rapid analysis of protein-protein interactions directly from DNA.

Journal Article Protein Sci · August 2011 Assays that integrate detection of binding with cell-free protein expression directly from DNA can dramatically increase the pace at which protein-protein interactions (PPIs) can be analyzed by mutagenesis. In this study, we present a method that combines ... Full text Link to item Cite

Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family.

Journal Article Cell · April 15, 2011 Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5' structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. W ... Full text Link to item Cite

A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity.

Journal Article Proteins · April 2011 Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing th ... Full text Link to item Cite

Programmable ligand detection system in plants through a synthetic signal transduction pathway.

Journal Article PLoS One · January 25, 2011 BACKGROUND: There is an unmet need to monitor human and natural environments for substances that are intentionally or unintentionally introduced. A long-sought goal is to adapt plants to sense and respond to specific substances for use as environmental mon ... Full text Link to item Cite

Thermodynamic analysis of ligand-induced changes in protein thermal unfolding applied to high-throughput determination of ligand affinities with extrinsic fluorescent dyes.

Journal Article Biochemistry · December 28, 2010 The quantification of protein-ligand interactions is essential for systems biology, drug discovery, and bioengineering. Ligand-induced changes in protein thermal stability provide a general, quantifiable signature of binding and may be monitored with dyes ... Full text Open Access Link to item Cite

Multifactorial determinants of protein expression in prokaryotic open reading frames.

Journal Article J Mol Biol · October 8, 2010 A quantitative description of the relationship between protein expression levels and open reading frame (ORF) nucleotide sequences is important for understanding natural systems, designing synthetic systems, and optimizing heterologous expression. Codon id ... Full text Link to item Cite

Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity.

Journal Article Proc Natl Acad Sci U S A · March 16, 2010 The Gibbs free energy difference between native and unfolded states ("stability") is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small mol ... Full text Link to item Cite

Structural analysis of semi-specific oligosaccharide recognition by a cellulose-binding protein of thermotoga maritima reveals adaptations for functional diversification of the oligopeptide periplasmic binding protein fold.

Journal Article J Biol Chem · November 27, 2009 Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instan ... Full text Link to item Cite

Structural adaptations that modulate monosaccharide, disaccharide, and trisaccharide specificities in periplasmic maltose-binding proteins.

Journal Article J Mol Biol · May 29, 2009 Periplasmic binding proteins comprise a superfamily that is present in archaea, prokaryotes, and eukaryotes. Periplasmic binding protein ligand-binding sites have diversified to bind a wide variety of ligands. Characterization of the structural mechanisms ... Full text Link to item Cite

Engineering key components in a synthetic eukaryotic signal transduction pathway.

Journal Article Mol Syst Biol · 2009 Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) ... Full text Link to item Cite

Structural analysis of a periplasmic binding protein in the tripartite ATP-independent transporter family reveals a tetrameric assembly that may have a role in ligand transport.

Journal Article J Biol Chem · November 21, 2008 Several bacterial solute transport mechanisms involve members of the periplasmic binding protein (PBP) superfamily that bind and deliver ligand to integral membrane transport proteins in the ATP-binding cassette, tripartite tricarboxylate transporter, or t ... Full text Link to item Cite

Ligand-induced conformational changes in a thermophilic ribose-binding protein.

Journal Article BMC Struct Biol · November 19, 2008 BACKGROUND: Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the ... Full text Link to item Cite

Structural reorganization and preorganization in enzyme active sites: comparisons of experimental and theoretically ideal active site geometries in the multistep serine esterase reaction cycle.

Journal Article J Am Chem Soc · November 19, 2008 Many enzymes catalyze reactions with multiple chemical steps, requiring the stabilization of multiple transition states during catalysis. Such enzymes must strike a balance between the conformational reorganization required to stabilize multiple transition ... Full text Link to item Cite

In the wake of two retractions, a request for investigation.

Journal Article Nature · July 24, 2008 Full text Link to item Cite

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

Journal Article BMC Struct Biol · March 28, 2008 BACKGROUND: Comparison of experimentally determined mesophilic and thermophilic homologous protein structures is an important tool for understanding the mechanisms that contribute to thermal stability. Of particular interest are pairs of homologous structu ... Full text Link to item Cite

Retraction.

Journal Article Science · February 1, 2008 Full text Link to item Cite

Structure-based design of robust glucose biosensors using a Thermotoga maritima periplasmic glucose-binding protein.

Journal Article Protein Sci · October 2007 We report the design and engineering of a robust, reagentless fluorescent glucose biosensor based on the periplasmic glucose-binding protein obtained from Thermotoga maritima (tmGBP). The gene for this protein was cloned from genomic DNA and overexpressed ... Full text Link to item Cite

Protein fabrication automation.

Journal Article Protein Sci · March 2007 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 p ... Full text Link to item Cite

Local encoding of computationally designed enzyme activity.

Journal Article J Mol Biol · February 23, 2007 One aim of computational protein design is to introduce novel enzyme activity into proteins of known structure by predicting mutations that stabilize transition states. Previously, we showed that it is possible to introduce triose phosphate isomerase activ ... Full text Link to item Cite

The crystal structure of a thermophilic glucose binding protein reveals adaptations that interconvert mono and di-saccharide binding sites.

Journal Article J Mol Biol · September 15, 2006 Periplasmic binding proteins (PBPs) comprise a protein superfamily that is involved in prokaryotic solute transport and chemotaxis. These proteins have been used to engineer reagentless biosensors to detect natural or non-natural ligands. There is consider ... Full text Link to item Cite

Binding and signaling of surface-immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins.

Journal Article Protein Sci · August 2006 Development of biosensor devices typically requires incorporation of the molecular recognition element into a solid surface for interfacing with a signal detector. One approach is to immobilize the signal transducing protein directly on a solid surface. He ... Full text Link to item Cite

Identification of cognate ligands for the Escherichia coli phnD protein product and engineering of a reagentless fluorescent biosensor for phosphonates.

Journal Article Protein Sci · July 2006 The Escherichia coli phnD gene is hypothesized to code for the periplasmic binding component of a phosphonate uptake system. Here we report the characterization of the phosphonate-binding properties of the phnD protein product. We find that PhnD exhibits h ... Full text Link to item Cite

Design of a calcium-binding protein with desired structure in a cell adhesion molecule.

Journal Article J Am Chem Soc · February 23, 2005 Ca2+, "a signal of life and death", controls numerous cellular processes through interactions with proteins. An effective approach to understanding the role of Ca2+ is the design of a Ca2+-binding protein with predicted structural and functional properties ... Full text Link to item Cite

Analysis of allosteric signal transduction mechanisms in an engineered fluorescent maltose biosensor.

Journal Article Protein Sci · February 2005 We previously reported the construction of a family of reagentless fluorescent biosensor proteins by the structure-based design of conjugation sites for a single, environmentally sensitive small molecule dye, thus providing a mechanism for the transduction ... Full text Link to item Cite

Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms.

Journal Article Protein Sci · January 2005 Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site-s ... Full text Link to item Cite

Periplasmic binding proteins: a versatile superfamily for protein engineering.

Journal Article Curr Opin Struct Biol · August 2004 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 a ... Full text Link to item Cite

Protein constructed to detect nerve agent weapons

Journal Article Industrial Bioprocessing · July 1, 2004 Researchers from Duke University constructed a protein to detect pinacolyl methyl phosphonic acid (PMPA). Binding sites of periplasmic binding proteins from Escherichia coli were redesigned to fit the PMPA molecule. The constructed proteins were tested for ... Cite

Computational design of a biologically active enzyme.

Journal Article Science · June 25, 2004 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 ha ... Full text Link to item Cite

Computational design of receptors for an organophosphate surrogate of the nerve agent soman.

Journal Article Proc Natl Acad Sci U S A · May 25, 2004 We report the computational design of soluble protein receptors for pinacolyl methyl phosphonic acid (PMPA), the predominant hydrolytic product of the nerve agent soman. Using recently developed computational protein design techniques, the ligand-binding p ... Full text Link to item Cite

Computational design of a Zn2+ receptor that controls bacterial gene expression.

Journal Article Proc Natl Acad Sci U S A · September 30, 2003 The control of cellular physiology and gene expression in response to extracellular signals is a basic property of living systems. We have constructed a synthetic bacterial signal transduction pathway in which gene expression is controlled by extracellular ... Full text Link to item Cite

Rational design of a calcium-binding protein.

Journal Article J Am Chem Soc · May 21, 2003 Calcium ions play key roles as structural components in biomineralization and as a second messenger in signaling pathways. We have introduced a de novo designed calcium-binding site into the framework of a non-calcium-binding protein, domain 1 of CD2. The ... Full text Link to item Cite

An empirical model for electrostatic interactions in proteins incorporating multiple geometry-dependent dielectric constants.

Journal Article Proteins · May 15, 2003 Featured Publication Here we introduce an electrostatic model that treats the complexity of electrostatic interactions in a heterogeneous protein environment by using multiple parameters that take into account variations in protein geometry, local structure, and the type of in ... Full text Link to item Cite

Computational design of receptor and sensor proteins with novel functions.

Journal Article Nature · May 8, 2003 Featured Publication 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 biote ... Full text Link to item Cite

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

Journal Article Nat Mater · November 2002 Featured Publication 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-ATP ... Full text Link to item Cite

Construction of a fluorescent biosensor family.

Journal Article Protein Sci · November 2002 Featured Publication Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism ... Full text Link to item Cite

Structural analysis, identification, and design of calcium-binding sites in proteins.

Journal Article Proteins · May 15, 2002 Assigning proteins with functions based on the 3-D structure requires high-speed techniques to make a systematic survey of protein structures. Calcium regulates many biological systems by binding numerous proteins in different biological environments. Desp ... Full text Link to item Cite

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

Journal Article Biochemistry · March 5, 2002 Featured Publication 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 oxyge ... Full text Link to item Cite

The crystal structure of human protein farnesyltransferase reveals the basis for inhibition by CaaX tetrapeptides and their mimetics.

Journal Article Proc Natl Acad Sci U S A · November 6, 2001 Protein farnesyltransferase (FTase) catalyzes the attachment of a farnesyl lipid group to the cysteine residue located in the C-terminal tetrapeptide of many essential signal transduction proteins, including members of the Ras superfamily. Farnesylation is ... Full text Link to item Cite

Manipulation of ligand binding affinity by exploitation of conformational coupling.

Journal Article Nat Struct Biol · September 2001 Featured Publication Traditional approaches for increasing the affinity of a protein for its ligand focus on constructing improved surface complementarity in the complex by altering the protein binding site to better fit the ligand. Here we present a novel strategy that leaves ... Full text Link to item Cite

Design of bioelectronic interfaces by exploiting hinge-bending motions in proteins.

Journal Article Science · August 31, 2001 Featured Publication We report a flexible strategy for transducing ligand-binding events into electrochemical responses for a wide variety of proteins. The method exploits ligand-mediated hinge-bending motions, intrinsic to the bacterial periplasmic binding protein superfamily ... Full text Link to item Cite

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

Journal Article Proc Natl Acad Sci U S A · April 24, 2001 Featured Publication 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 f ... Full text Link to item Cite

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

Journal Article J Mol Biol · March 16, 2001 Featured Publication 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 ... Full text Link to item Cite

Synthesis and characterization of a ruthenium(II)-based redox conjugate for reagentless biosensing.

Journal Article Bioconjug Chem · 2001 Synthesis of a novel sulfhydryl-specific, tetraammine Ru(II)polypyridyl complex, [Ru(II)(NH(3))(4)(1,10-phenanthroline-5-maleimide)](PF(6))(2), which exhibits environment-sensitive electrochemical properties is described. When conjugated to an allosteric s ... Full text Link to item Cite

Rational design of nascent metalloenzymes.

Journal Article Proc Natl Acad Sci U S A · June 6, 2000 Featured Publication 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 ... Full text Link to item Cite

Dissection of the protein G B1 domain binding site for human IgG Fc fragment.

Journal Article Protein Sci · August 1999 The contribution to the free energy of binding of each of the residues forming the binding site for a human IgG Fc fragment on the surface of the B1 domain of protein G was determined by alanine-scanning mutagenesis. The interface between these two protein ... Full text Link to item Cite

Construction of a blue copper analogue through iterative rational protein design cycles demonstrates principles of molecular recognition in metal center formation

Journal Article Journal of the American Chemical Society · October 7, 1998 The construction and characterization of a series of proteins in which the Blue Copper CysHis2Met primary coordination sphere was placed in various orientations within the hydrophobic core of thioredoxin has allowed exploration of the principles of molecul ... Full text Cite

Structure-based engineering of environmentally sensitive fluorophores for monitoring protein-protein interactions.

Journal Article Protein Eng · September 1998 Single, extrinsic, environmentally sensitive fluorophores can be used to quantitate formation of protein-protein complexes. These can be prepared semi-synthetically by covalent coupling to single cysteine mutations introduced at positions where the fluorop ... Full text Link to item Cite

The development of new biotechnologies using metalloprotein design.

Journal Article Curr Opin Biotechnol · August 1998 A variety of methodologies are under development to alter the behavior of existing metal centers or create entirely new sites within a protein framework in order to exploit the intrinsic chemical versatility of metals using the exquisite level of control t ... Full text Link to item Cite

Computational protein engineering.

Journal Article Nat Struct Biol · July 1998 Full text Link to item Cite

Construction of a family of Cys2His2 zinc binding sites in the hydrophobic core of thioredoxin by structure-based design.

Journal Article Biochemistry · June 9, 1998 Featured Publication A semi-automated, rational design strategy has been used to introduce a family of seven single, mononuclear Cys2His2 zinc sites at various locations in the hydrophobic core of Escherichia colithioredoxin, a protein that is normally devoid of metal centers. ... Full text Link to item Cite

Construction of a novel redox protein by rational design: conversion of a disulfide bridge into a mononuclear iron-sulfur center.

Journal Article Biochemistry · May 19, 1998 Featured Publication A mononuclear iron-sulfur center, capable of reversible electron transfer, has been introduced into thioredoxin, a protein devoid of such sites, using an automated, structure-based design algorithm. One of the sites predicted by the Dezymer computer progra ... Full text Link to item Cite

Protein engineering and the development of generic biosensors.

Journal Article Trends Biotechnol · April 1998 Featured Publication Biosensors exploit the remarkable specificity of biomolecular recognition to provide analytical tools that can measure the presence of a single molecular species in a complex mixture. A new strategy is emerging in the development of biosensor technologies: ... Full text Link to item Cite

Genetic engineering of a single-chain antibody fragment for surface immobilization in an optical biosensor.

Journal Article Biosens Bioelectron · March 1, 1998 The development of a biosensor based on a genetically engineered biomolecule offers many potential advantages to sensors that rely on natural proteins only. Here we present how protein engineering techniques can be used to introduce a functional unit for s ... Full text Link to item Cite

Engineering biosensors by introducing fluorescent allosteric signal transducers: Construction of a novel glucose sensor

Journal Article Journal of the American Chemical Society · January 14, 1998 The development of biosensors based on genetically engineered proteins offers many potential advantages to sensors that rely on natural proteins only. Here we present how protein engineering techniques can be used to integrate optical signal transduction f ... Full text Cite

The construction of metal centers in proteins by rational design.

Journal Article Fold Des · 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 protei ... Full text Link to item Cite

Rational protein design: combining theory and experiment.

Journal Article Proc Natl Acad Sci U S A · September 16, 1997 Full text Link to item Cite

The rational design and construction of a cuboidal iron-sulfur protein.

Journal Article Proc Natl Acad Sci U S A · June 24, 1997 Featured Publication Rational protein design is an emerging approach for testing general theories of protein chemistry through the creation of new structures and functions. Here we present the first successful introduction by rational design of a [Fe4S4] cuboidal cluster into ... Full text Link to item Cite

Construction of a catalytically active iron superoxide dismutase by rational protein design.

Journal Article Proc Natl Acad Sci U S A · May 27, 1997 Featured Publication The rational protein design algorithm DEZYMER was used to introduce the active site of nonheme iron superoxide dismutase (SOD) into the hydrophobic interior of the host protein, Escherichia coli thioredoxin (Trx), a protein that does not naturally contain ... Full text Link to item Cite

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

Journal Article Proc Natl Acad Sci U S A · April 29, 1997 Featured Publication 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 app ... Full text Link to item Cite

NMR studies of structure, hydrogen exchange, and main-chain dynamics in a disrupted-core mutant of thioredoxin.

Journal Article Protein Sci · December 1996 Core-packing mutants of proteins often approach molten globule states, and hence may have attributes of folding intermediates. We have studied a core-packing mutant of thioredoxin, L78K, in which a leucine residue is substituted by lysine, using 15N hetero ... Full text Link to item Cite

Metalloprotein design.

Journal Article Curr Opin Biotechnol · August 1996 The rational design of novel proteins offers a new method of studying structure and function, and makes possible the construction of new biomaterials. The richness of metal chemistry, the relative ease of creating stable complexes, and the remarkable degre ... Full text Link to item Cite

Mapping staphylococcal nuclease conformation using an EDTA-Fe derivative attached to genetically engineered cysteine residues.

Journal Article Biochemistry · November 22, 1994 Six single cysteine variants of staphylococcal nuclease were reacted with the iron complex of (EDTA-2-aminoethyl) 2-pyridyl disulfide (EPD-Fe) [Ermácora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., & Fox, R. O. (1992) Proc. Natl. Acad. Sci. U.S.A. 8 ... Full text Link to item Cite

Optimal sequence selection in proteins of known structure by simulated evolution.

Journal Article Proc Natl Acad Sci U S A · June 21, 1994 Featured Publication Rational design of protein structure requires the identification of optimal sequences to carry out a particular function within a given backbone structure. A general solution to this problem requires that a potential function describing the energy of the s ... Full text Link to item Cite

Thermodynamic effects of reduction of the active-site disulfide of Escherichia coli thioredoxin explored by differential scanning calorimetry.

Journal Article Biochemistry · March 29, 1994 Intramolecular disulfide bonds in protein molecules, whether present in the wild-type protein or engineered via site-directed mutagenesis, are capable of significantly increasing the stability. Establishing thermodynamic parameters associated with the redo ... Full text Link to item Cite

Stability of oxidized Escherichia coli thioredoxin and its dependence on protonation of the aspartic acid residue in the 26 position.

Journal Article Biochemistry · July 27, 1993 The effects of pH in the range 6.0-8.0 on the thermodynamics of the reversible thermal unfolding of Escherichia coli thioredoxin in the oxidized state have been determined over a range of concentrations using differential scanning calorimetry. The thermal ... Full text Link to item Cite

Integrated optical waveguide attenuated total reflection spectrometry and resonance Raman spectroscopy of adsorbed cytochrome c

Journal Article Journal of Physical Chemistry · January 1, 1993 The heme group of cytochrome c (Cyt C) has resonance absorptions at 520 and 550 nm that arise from x-y degenerate in-plane electronic transitions of the heme moiety. In the present paper, horse heart Cyt C was adsorbed to the surface of a micron-thick sili ... Full text Cite

The hydrophobic core of Escherichia coli thioredoxin shows a high tolerance to nonconservative single amino acid substitutions.

Journal Article Biochemistry · November 17, 1992 A set of single amino acid substitutions has been constructed at positions Leu42 and Leu78 in the hydrophobic core of Escherichia coli thioredoxin. This protein is required for the in vivo assembly of filamentous bacteriophages such as M13. Almost all the ... Full text Link to item Cite

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

Journal Article J Mol Biol · December 5, 1991 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 ba ... Full text Link to item Cite

Construction of new ligand binding sites in proteins of known structure. II. Grafting of a buried transition metal binding site into Escherichia coli thioredoxin.

Journal Article J Mol Biol · December 5, 1991 In an accompanying paper a computational procedure is described, which introduces new ligand-binding sites into proteins of known structure. Here we describe the experimental implementation of one of the designs, which is intended to introduce a copper-bin ... Full text Link to item Cite

Site-directed mutagenesis in the effector site of Escherichia coli phosphofructokinase.

Journal Article Biochemistry · June 30, 1987 A new vector for the expression of phosphofructokinase (pfk-1) was constructed with pEMBL, which allows reliable, inducible, high-expression, and facile mutagenesis of the gene. Two mutants in the effector site of the enzyme were produced by site-specific ... Full text Link to item Cite

Mutations in the active site of Escherichia coli phosphofructokinase.

Journal Article Nature · June 4, 1987 The enzyme-catalysed transfer of a phosphoryl group from ATP is an important reaction in a wide variety of biological processes. We demonstrate here the essential function of an aspartate group in the catalysis of phosphoryl transfer by Escherichia coli ph ... Full text Link to item Cite

Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase.

Journal Article Eur J Biochem · June 3, 1985 The gene for the major phosphofructokinase enzyme in Escherichia coli, pfkA, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle e ... Full text Link to item Cite