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

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, to establish allosterically controlled interactions between electrode surfaces and redox-active, Ru(II)-labeled proteins. This approach allows the development of protein-based bioelectronic interfaces that respond to a diverse set of analytes. Families of these interfaces can be generated either by exploiting natural binding diversity within the superfamily or by reengineering the specificity of individual proteins. These proteins may have numerous medical, environmental, and defense applications.

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

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.

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

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.

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

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-binding site into Escherichia coli thioredoxin. The new binding site can be introduced with a minimum of four amino acid changes. The binding site is buried so that structural rules for making mutations in the hydrophobic core of a protein, as well as for the introduction of new functions, are being tested in this experiment. The mutant protein is folded even in the absence of metals, and variants that retain the original activity of thioredoxin can be isolated. The protein has gained a metal-binding site specific for transition metals. The metal co-ordination chemistry at the binding site varies depending on the metal that is introduced into it. Mercury(II) is co-ordinated in the expected manner. Copper(II) binds in a way that was not anticipated in the original design. It appears to use two of the four residues intended to form the co-ordination sphere, and two other residues that were not part of the original set of mutations. It is therefore necessary not only to introduce new functional groups to form a new site, but also to consider and remove alternative modes of binding.

Metalloprotein design.

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 degree of subtle, highly specific control of reactivity imposed by the protein matrix upon the metal center make metalloprotein design a very fruitful area for the exploration and application of design techniques. So far, most designs have concentrated on the exploration of simple metal-chelation properties. Even so, this has led to the development of new methods for protein stabilization and affinity purification, of metal biosensors, of novel strategies for control of protein activity, and of model systems for the exploration of fundamental principles of molecular recognition.

Protein engineering and the development of generic biosensors.

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: molecular-engineering techniques are being used to adapt the properties of proteins to simple, generic detector instrumentation, rather than adapting instruments to the unique requirements of a natural molecule.

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

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 proteins is atypical in that it is smaller than usual, polar in character, and involves two well-defined "knobs-into-holes" interactions. The bulk of the free energy of binding is contributed by three central residues, which make hydrogen bonds across the interface. Of these, the most critical interaction is formed by Glu27, which acts as a charged knob on the surface of the B1 domain, inserting into a polar hole on the Fc fragment. A single alanine mutation of this residue virtually abolishes stable complex formation. Formation of a stable interface between these two proteins is therefore dominated by a small, polar "hot spot."

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

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 heteronuclear two- and three-dimensional NMR. Chemical shift differences between the mutant and wild-type main-chain resonances reveal that structural changes caused by the mutation are localized within 12 A of the altered side chain. The majority of resonances are unchanged, as are many 1H-1H NOEs indicative of the main-chain fold, suggesting that the structure of L78K is largely similar to wild type. Hydrogen exchange studies reveal that residues comprising the central beta-sheet of both mutant and wild-type proteins constitute a local unfolding unit, but with the unfolding/folding equilibrium approximately 12 times larger in L78K. The dynamics of main-chain NH bonds in L78K were studied by 15N spin relaxation and compared with a previous study of wild type. Order parameters for angular motion of NH bonds in the mutant are on average lower than in wild type, suggesting greater spatial freedom on a rapid time scale, but may also be related to different rotational correlation times in the two proteins. There is also evidence of greater conformational exchange in the mutant. Differences between mutant and wild type in hydrogen exchange and main-chain dynamics are not confined to the vicinity of the mutation. We infer that mispacking of the protein core in one location affects local dynamics and stability throughout.

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

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 surface immobilization into a single-chain antibody fragment (scFv). A peptide known to mimic the binding properties of biotin was fused to the carboxyterminus of the phosphorylcholine-binding scFv fragment of IgA McPC603. This fusion protein could be immobilized on a streptavidin monolayer. The resulting scFv monolayer was capable of binding a fluorescently labeled phosphorylcholine analog, as detected by total internal reflection fluorescence. In contrast, an scFv monolayer formed by introducing biotin through chemical modification was not capable of binding phosphorylcholine. These results demonstrate the utility of site-specific, oriented attachment strategies in the formation of protein monolayers in optical sensors, made possible by the use of protein engineering techniques.

The construction of metal centers in proteins by rational design.

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.

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

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 system as a function of its atomic coordinates be minimized simultaneously over all available sequences and their three-dimensional atomic configurations. Here we present a method that explicitly minimizes a semiempirical potential function simultaneously in these two spaces, using a simulated annealing approach. The method takes the fixed three-dimensional coordinates of a protein backbone and stochastically generates possible sequences through the introduction of random mutations. The corresponding three-dimensional coordinates are constructed for each sequence by "redecorating" the backbone coordinates of the original structure with the corresponding side chains. These are then allowed to vary in their structure by random rotations around free torsional angles to generate a stochastic walk in configurational space. We have named this method protein simulated evolution, because, in loose analogy with natural selection, it randomly selects for allowed solutions in the sequence of a protein subject to the "selective pressure" of a potential function. Energies predicted by this method for sequences of a small group of residues in the hydrophobic core of the phage lambda cI repressor correlate well with experimentally determined biological activities. This "genetic selection by computer" approach has potential applications in protein engineering, rational protein design, and structure-based drug discovery.

Manipulation of ligand binding affinity by exploitation of conformational coupling.

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 the binding site intact, while residues that allosterically affect binding are mutated. This method takes advantage of conformationally distinct states, each with different ligand-binding affinities, and manipulates the equilibria between these conformations. We demonstrate this approach in the Escherichia coli maltose binding protein by introducing mutations, located at some distance from the ligand binding pocket, that sterically affect the equilibrium between an open, apo-state and a closed, ligand-bound state. A family of 20 variants was generated with affinities ranging from an approximately 100-fold improvement (7.4 nM) to an approximately two-fold weakening (1.8 mM) relative to the wild type protein (800 nM).

Mutations in the active site of Escherichia coli phosphofructokinase.

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 phosphofructokinase, and the minor role of an arginine residue. We have used oligonucleotide-directed mutagenesis to replace two amino-acid residues which X-ray analysis has shown to be close to the transferred phosphoryl group and we have analysed the forward and back reactions of the mutant enzymes by steady-state kinetics. Changing Asp 127 to Ser reduced the turnover number by a factor of 18,000 in the forward direction and 3,100 in the back reaction, and the Michaelis constant for fructose 1,6-bisphosphate in the reverse reaction by a factor of 45. This shows that this aspartate is a key residue in the rate enhancement by the enzyme, probably acting as a base in the reaction mechanism, and that it also destabilizes the product complex. Changing Arg 171 to Ser reduced the turnover numbers by about 3.4, showing that this arginine has only a minor effect on the catalysis.

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

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 enzymes, but is different from the second, minor phosphofructokinase found in E. coli. The region which has been sequenced comprises the complete pfkA--tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate-binding protein, sbp, and for a membrane-bound enzyme, CDP-diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA-sbp-cdh-tpi. The pfkA gene has been subcloned into a high-copy-number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 X 10(3)-nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40-50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using 'reverse genetics'.

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

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.

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

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 fluorophore is predicted to respond to formation of the complex without adversely affecting the interaction. The three-dimensional structure of a protein-protein interface can be used to select such locations by identifying residues that are located at the edge of a buried interfacial region, and are in partial steric contact with both partners as indicated by a change in their static solvent-accessible surface area upon complex formation. Using this design approach, cysteine mutations were introduced into the B1 domain of protein G, which successfully monitor complex formation with minimal interference. Such constructs have great utility in the analysis of solution properties of interface mutants.

Rational design of nascent metalloenzymes.

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.

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

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 Zn2+. In this system a computationally designed Zn2+-binding periplasmic receptor senses the extracellular solute and triggers a two-component signal transduction pathway via a chimeric transmembrane protein, resulting in transcriptional up-regulation of a beta-galactosidase reporter gene. The Zn2+-binding site in the designed receptor is based on a four-coordinate, tetrahedral primary coordination sphere consisting of histidines and glutamates. In addition, mutations were introduced in a secondary coordination sphere to satisfy the residual hydrogen-bonding potential of the histidines coordinated to the metal. The importance of the secondary shell interactions is demonstrated by their effect on metal affinity and selectivity, as well as protein stability. Three designed protein sequences, comprising two distinct metal-binding positions, were all shown to bind Zn2+ and to function in the cell-based assay, indicating the generality of the design methodology. These experiments demonstrate that biological systems can be manipulated with computationally designed proteins that have drastically altered ligand-binding specificities, thereby extending the repertoire of genetic control by extracellular signals.

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

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 the hydrophobic core of Escherichia coli thioredoxin, a protein normally devoid of metal centers. Cuboidal [Fe4S4] is one of the stable forms of self-assembled iron-sulfur clusters that are thought to represent some of the earliest evolved biological redox centers. [Fe4S4] clusters have been recruited for use in a variety of proteins whose functions are central to many of the major biochemical processes ranging from simple soluble electron-transfer agents, to membrane-bound components of electron-transfer chains, to electron reservoirs in complex metalloenzymes such as nitrogenase. By situating an [Fe4S4] cluster into a protein environment not previously adapted by evolution we can explore the factors by which their activity is modulated by the protein matrix.

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

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 a transition metal-binding site. Reconstitution of the designed protein, Trx-SOD, showed the incorporation of one high-affinity metal-binding site. The electronic spectra of the holoprotein and its N3- and F- adducts are analogous to those previously reported for native {Fe3+}SOD. Activity assays showed that {Fe3+}Trx-SOD is capable of catalyzing the dismutation of the superoxide anion; comparative studies with the unrelated wild-type E. coli iron SOD indicated that {Fe3+}Trx-SOD catalyzes the dismutation reaction at a rate on the order of 10(5) M-1s -1. The ability to design catalytically competent metalloenzymes allows for the systematic investigation of fundamental mechanistic questions concerning catalysis at transition metal centers.