Non-contiguous protein recombination

Swapping sequence elements among related proteins can produce chimeric proteins with novel behaviors and improved properties such as enhanced stability. Although homologous mutations are much more conservative than random mutations, chimeras of distantly-related proteins have a low probability of retaining fold and function. Here, I introduce a new tool for protein recombination that identifies structural blocks that can be swapped among homologous proteins with minimal disruption. This non-contiguous recombination approach enables design of chimeras and libraries of chimeras with less disruption than can be achieved by swapping blocks of sequence. Less disruption means that one can generate libraries with higher fractions of functional enzymes and enables recombination of more distant homologs.

Using this new tool I design and construct many functional chimeric cellulases. I illustrate the structurally conservative nature of this recombination by creating a functional prokaryotic-eukaryotic chimera and solving its structure. I also show how non-contiguous recombination can be used to efficiently identify stabilizing mutations that have been incorporated into homologs in nature.

Structural and mechanistic motifs in membrane proteins: the three-dimensional modelling of rhodopsin, band 3, and the nicotinic acetylcholine receptor

Because so little is known about the structure of membrane proteins, an attempt has been made in this work to develop techniques by which to model them in three dimensions. The procedures devised rely heavily upon the availability of several sequences of a given protein. The modelling procedure is composed of two parts. The first identifies transmembrane regions within the protein sequence on the basis of hydrophobicity, β-turn potential, and the presence of certain amino acid types, specifically, proline and basic residues. The second part of the procedure arranges these transmembrane helices within the bilayer based upon the evolutionary conservation of their residues. Conserved residues are oriented toward other helices and variable residues are positioned to face the surrounding lipids. Available structural information concerning the protein's helical arrangement, including the lengths of interhelical loops, is also taken into account. Rhodopsin, band 3, and the nicotinic acetylcholine receptor have all been modelled using this methodology, and mechanisms of action could be proposed based upon the resulting structures.

Specific residues in the rhodopsin and iodopsin sequences were identified, which may regulate the proteins' wavelength selectivities. A hinge-like motion of helices M3, M4, and M5 with respect to the rest of the protein was proposed to result in the activation of transducin, the G-protein associated with rhodopsin. A similar mechanism is also proposed for signal transduction by the muscarinic acetylcholine and β-adrenergic receptors.

The nicotinic acetylcholine receptor was modelled with four trans-membrane helices per subunit and with the five homologous M2 helices forming the cation channel. Putative channel-lining residues were identified and a mechanism of channel-opening based upon the concerted, tangential rotation of the M2 helices was proposed.

Band 3, the anion exchange protein found in the erythrocyte membrane, was modelled with 14 transmembrane helices. In general the pathway of anion transport can be viewed as a channel composed of six helices that contains a single hydrophobic restriction. This hydrophobic region will not allow the passage of charged species, unless they are part of an ion-pair. An arginine residue located near this restriction is proposed to be responsible for anion transport. When ion-paired with a transportable anion it rotates across the barrier and releases the anion on the other side of the membrane. A similar process returns it to its original position. This proposed mechanism, based on the three-dimensional model, can account for the passive, electroneutral, anion exchange observed for band 3. Dianions can be transported through a similar mechanism with the additional participation of a histidine residue. Both residues are located on M10.

Part I. Synchronization of the cell division cycle of HeLa cells in suspension cultures. Part II. Studies on chromosomal proteins of HeLa cells during the cell division cycle

Part I

These studies investigate the potential of single and double treatments with either 5-fluorodeoxyuridine of excess thymidine to induce cell division synchrony in suspension cultures of HeLa cells. The patterns of nucleic acid synthesis and cell proliferation have been analyzed in cultures thus synchronized. Several changes in cell population during long incubation with 5-fluorodeoxyuridine or excess thymidine are also described. These results are subjected to detailed evaluation in terms of the degree and quality of synchrony finally achieved.

Part II

Histones and non-histone proteins associated with interphase and metaphase chromosomes of HeLa cells have been qualitatively and quantitatively analyzed. Histones were fractionated by chromatography on Amberlite CG-50 and further characterized by analytical disc electrophoresis and amino acid analysis of each chromatographic fraction. It is concluded that histones of HeLa cells are comprised of only a small number of major components and that these components are homologous to those of other higher organisms. Of all the histones, arginine-rich histone III alone contains cysteine and can polymerize through formation of intermolecular disulfide bridges between histone III monomers.

A detailed comparison by chromatography and disc electrophoresis established that interphase and metaphase histones are made up of similar components. However, certain quantitative differences in proportions of different histones of interphase and metaphase cells are reported. Indirect evidence indicates that a certain proportion of metaphase histone III is polymerized through intermolecular disulfide links, whereas interphase histone III occurs mainly in the monomeric form.

Metaphase chromosomes are associated with an additional acid-soluble protein fraction which is absent from interphase chromosomes. All of these additional acid-soluble proteins of metaphase chromosomes are shown to be non-histones and it is concluded that the histone/DNA ratio is identical in interphase and metaphase chromosomes. The bulk of acid-soluble non-histone proteins of metaphase chromosomes were found to be polymerized through disulfide bridges; corresponding interphase non-histone proteins displayed no evidence of similar polymerization.

The factors responsible for the condensed configuration and metabolic inactivity of metaphase chromosomes are discussed in light of these findings.

The relationship between histone and DNA synthesis in nondividing differentiated chicken erythrocyte cells and in rapidly dividing undifferentiated HeLa cells is also investigated. Of all the histones, only arginine-rich histones are synthesized in mature erythrocytes. Histone synthesis in HeLa cells was studied in both unsynchronized and synchronized cultures. In HeLa cells, only part of the synthesis of all histone fractions is dependent on concurrent DNA synthesis, whereas all histones are synthesized in varying degrees even in the absence of DNA synthesis.

Pharmacology and pore-forming domains of the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel member of the ATP-binding cassette (ABC) superfamily of membrane proteins. CFTR has two homologous halves, each consisting of six transmembrane spanning domains (TM) followed by a nucleotide binding fold, connected by a regulatory (R) domain. This thesis addresses the question of which domains are responsible for Cl^- selectivity, i.e., which domains line the channel pore.

To address this question, novel blockers of CFTR were characterized. CFTR was heterologously expressed in Xenopus oocytes to study the mechanism of block by two closely related arylaminobenzoates, diphenylamine-2-carboxylic acid (DPC) and flufenamic acid (FFA). Block by both is voltage-dependent, with a binding site ≈ 40% through the electric field of the membrane. DPC and FFA can both reach their binding site from either side of the membrane to produce a flickering block of CFTR single channels. In addition, DPC block is influenced by Cl^- concentration, and DPC blocks with a bimolecular forward binding rate and a unimolecular dissociation rate. Therefore, DPC and FFA are open-channel blockers of CFTR, and a residue of CFTR whose mutation affects their binding must line the pore.

Screening of site-directed mutants for altered DPC binding affinity reveals that TM-6 and TM-12 line the pore. Mutation of residue 5341 in TM-6 abolishes most DPC block, greatly reduces single-channel conductance, and alters the direction of current rectification. Additional residues are found in TM-6 (K335) and TM-12 (T1134) whose mutations weaken or strengthen DPC block; other mutations move the DPC binding site from TM-6 to TM-12. The strengthened block and lower conductance due to mutation T1134F is quantitated at the single-channel level. The geometry of DPC and of the residues mutated suggest α-helical structures for TM-6 and TM-12. Evidence is presented that the effects of the mutations are due to direct side-chain interaction, and not to allosteric effects propagated through the protein. Mutations are also made in TM-11, including mutation S1118F, which gives voltage-dependent current relaxations. The results may guide future studies on permeation through ABC transporters and through other Cl^- channels.

Identification of fucose-α(1-2)-galactose binding proteins in the mammalian brain

Fucose-α(1-2)-galactose (Fucα(1-2)Gal) carbohydrates have been implicated in cognitive functions. However, the underlying molecular mechanisms that govern these processes are not well understood. While significant progress has been made toward identifying glycoconjugates bearing this carbohydrate epitope, a major challenge remains the discovery of interactions mediated by these sugars. Here, we employ the use of multivalent glycopolymers to enable the proteomic identification of weak affinity, low abundant Fucα(1-2)Gal-binding proteins (i.e. lectins) from the brain. End-biotinylated glycopolymers containing photoactivatable crosslinkers were used to capture and enrich potential Fucα(1-2)Gal-specific lectins from rat brain lysates. Candidate lectins were tested for their ability to bind Fucα(1-2)Gal, and the functional significance of the interaction was investigated for one such candidate, SV2a, using a knock-out mouse system. Our results suggest an important role for this glycan-lectin interaction in facilitating synaptic changes necessary for neuronal communication. This study highlights the use of glycopolymer mimetics to discover novel lectins and identify functional interactions between fucosyl carbohydrates and lectins in the brain.

Engineering multi-step electron tunneling systems in proteins

Multi-step electron tunneling, or “hopping,” has become a fast-developing research field with studies ranging from theoretical modeling systems, inorganic complexes, to biological systems. In particular, the field is exploring hopping mechanisms in new proteins and protein complexes, as well as further understanding the classical biological hopping systems such as ribonuclease reductase, DNA photolyases, and photosystem II. Despite the plethora of natural systems, only a few biologically engineered systems exist. Engineered hopping systems can provide valuable information on key structural and electronic features, just like other kinds of biological model systems. Also, engineered systems can harness common biologic processes and utilize them for alternative reactions. In this thesis, two new hopping systems are engineered and characterized.

The protein Pseudomonas aeruginosa azurin is used as a building block to create the two new hopping systems. Besides being well studied and amenable to mutation, azurin already has been used to successfully engineer a hopping system. The two hopping systems presented in this thesis have a histidine-attached high potential rhenium 4,7-dimethyl-1,10-phenanthroline tricarbonyl [Re(dmp)(CO)3] + label which, when excited, acts as the initial electron acceptor. The metal donor is the type I copper of the azurin protein. The hopping intermediates are all tryptophan, an amino acid mutated into the azurin at select sites between the photoactive metal label and the protein metal site. One system exhibits an inter-molecular hopping through a protein dimer interface; the other system undergoes intra-molecular multi-hopping utilizing a tryptophan “wire.” The electron transfer reactions are triggered by excitation of the rhenium label and monitored by UV-Visible transient absorption, luminescence decays measurements, and time-resolved Infrared spectroscopy (TRIR). Both systems were structurally characterized by protein X-ray crystallography.

Isolation and characterization of proteins that bind to yeast origins of DNA replication

Yeast chromosomes contain sequences called ARSs which function as origins of replication in vitro and in vivo. We have carried out a systematic deletion analysis of ARS1, allowing us to define three functionally distinct domains, designated A, B, and C. Domain A is a sequence of 11 to 19bp, containing the core consensus element that is required for replication. The core consensus sequence, A/TTTTATPuTTTA/T, is conserved at all ARSs sequenced to date. A fragment containing only element A and 8 flanking nucleotides enables autonomous replication of centromeric plasmids. These plasmids replicate very inefficiently, suggesting that flanking sequences must be important for ARS function. Domain B also provides important sequences needed for efficient replication. Deletion of domain B drastically increases the doubling times of transformants and reduces plasmid stability. Domain B contains a potential consensus sequence conserved at some ARSs which overlaps a region of bent DNA. Mutational analysis suggests this bent DNA may be important for ARS function. Deletion of domain C has only a slight effect on replication of plasmids carrying those deletions.

We have identified a protein called ARS binding factor I (ABF-I) that binds to the HMR-E ARS and ARS1. We have purified this protein to homogeneity using conventional and oligonucleotide affinity chromatography. The protein has an apparent molecular weight of 135kDa and is present at about 700 molecules per diploid cell, based on the yield of purified protein and in situ antibody staining. DNaseI footprinting reveals that ABF-I binds sequence-specifically to an approximately 24bp sequence that overlaps element Bat ARSl. This same protein binds to and protects a similar size region at the HMR-E ARS.

We also find evidence for another ARS binding protein, ABF-III, based on DN asei footprint analysis and gel retardation assays. The protein protects approximately 22bp adjacent to the ABF-I site. There appears to be no interaction between ABF-I and ABF-III despite the proximity of their binding sites.

To address the function of ABF-I in DNA replication, we have cloned the ABF-I gene using rabbit polyclonal anti-sera and murine monoclonal antibodies against ABF-I to screen a λgt11 expression library. Four EcoRI restriction fragments were isolated which encoded proteins that were recognized by both polyclonal and monoclonal antibodies. A gene disruption can now be constructed to determine the in vivo function of ABF-I.

Revealing, illuminating, and modifying proteins in human diseases using noncanonical amino acids

To better understand human diseases, much recent work has focused on proteins to either identify disease targets through proteomics or produce therapeutics via protein engineering. Noncanonical amino acids (ncAAs) are tools for altering the chemical and physical properties of proteins, providing a facile strategy not only to label proteins but also to engineer proteins with novel properties. My thesis research has focused on the development and applications of noncanonical amino acids in identifying, imaging, and engineering proteins for studying human diseases. Chapter 1 introduces the concept of ncAAs and reveals insights to how I chose my thesis projects.

ncAAs have been incorporated to tag and enrich newly synthesized proteins for mass spectrometry through a method termed BONCAT, or bioorthogonal noncanonical amino acid tagging. Chapter 2 describes the investigation of the proteomic response of human breast cancer cells to induced expression of tumor suppressor microRNA miR-126 by combining BONCAT with another proteomic method, SILAC or stable isotope labeling by amino acids in cell culture. This proteomic analysis led to the discovery of a direct target of miR-126, shedding new light on its role in suppressing cancer metastasis.

In addition to mass spectrometry, ncAAs can also be utilized to fluorescently label proteins. Chapter 3 details the synthesis of a set of cell-permeant cyclooctyne probes and demonstration of selective labeling of newly synthesized proteins in live mammalian cells using azidohomoalanine. Similar to live cell imaging, the ability to selectively label a particular cell type within a mixed cell population is important to interrogating many biological systems, such as tumor microenvironments. By taking advantage of the metabolic differences between cancer and normal cells, Chapter 5 discusses efforts to develop selective labeling of cancer cells using a glutamine analogue.

Furthermore, Chapter 4 describes the first demonstration of global replacement at polar amino acid positions and its application in developing an alternative PEGylation strategy for therapeutic proteins. Polar amino acids typically occupy solvent-exposed positions on the protein surface, and incorporation of noncanonical amino acids at these positions should allow easier modification and cause less perturbation compared to replacements at the interior positions of proteins.

Characterization and developmental regulation of a gene expressed specifically in the skeletogenic lineage of the sea urchin embryo

The sea urchin embryonic skeleton, or spicule, is deposited by mesenchymal progeny of four precursor cells, the micromeres, which are determined to the skeletogenic pathway by a process known as cytoplasmic localization. A gene encoding one of the major products of the skeletogenic mesenchyme, a prominent 50 kD protein of the spicule matrix, has been characterized in detail. cDNA clones were first isolated by antibody screening of a phage expression library, followed by isolation of homologous genomic clones. The gene, known as SM50, is single copy in the sea urchin genome, is divided into two exons of 213 and 1682 bp, and is expressed only in skeletogenic cells. Transcripts are first detectable at the 120 cell stage, shortly after the segregation of the skeletogenic precursors from the rest of the embryo. The SM50 open reading frame begins within the first exon, is 450 amino acids in length, and contains a loosely repeated 13 amino acid motif rich in acidic residues which accounts for 45% of the protein and which is possibly involved in interaction with the mineral phase of the spicule.

The important cis-acting regions of the SM50 gene necessary for proper regulation of expression were identified by gene transfer experiments. A 562 bp promoter fragment, containing 438 bp of 5' promoter sequence and 124 bp of the SM50 first exon (including the SM50 initiation codon), was both necessary and sufficient to direct high levels of expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene specifically in the skeletogenic cells. Removal of promoter sequences between positions -2200 and -438, and of transcribed regions downstream of +124 (including the SM50 intron), had no effect on the spatial or transcriptional activity of the transgenes.

Regulatory proteins that interact with the SM50 promoter were identified by the gel retardation assay, using bulk embryo mesenchyme blastula stage nuclear proteins. Five protein binding sites were identified and mapped to various degrees of resolution. Two sites are homologous, may be enhancer elements, and at least one is required for expression. Two additional sites are also present in the promoter of the aboral ectoderm specific cytoskeletal actin gene CyIIIa; one of these is a CCAA T element, the other a putative repressor element. The fifth site overlaps the binding site of the putative repressor and may function as a positive regulator by interfering with binding of the repressor. All of the proteins are detectable in nuclear extracts prepared from 64 cell stage embryos, a stage just before expression of SM50 is initiated, as well as from blastula and gastrula stage; the putative enhancer binding protein may be maternal as well.

Electrochemical and electron transfer investigations of copper proteins

A study of the pH and temperature dependence of the redox potentials of azurins from five species of bacteria has been performed. The variations in the potentials with pH have been interpreted in terms of electrostatic interactions between the copper site and titrating histidine residues, including the effects of substitutions in the amino acid sequences of the proteins on the electrostatic interactions. A comparison of the observed pH dependences with predictions based on histidine pK_a values known for Pseudomonas aeruginosa (Pae), Alcaligenes denitrificans (Ade), and Alcaligenes faecalis (Afa) azurins indicates that the Pae and Ade redox potentials exhibit pH dependences in line with electrostatic arguments, while Afa azurin exhibits more complex behavior. Redox enthalpies and entropies for four of the azurins at low and high pH values have also been obtained. Based on these results in conjuction with the variable pH experiments, it appears that Bordetella bronchiseptica azurin may undergo a more substantial conformational change with pH than has been observed for other species of azurin.

The temperature dependence of the redox potential of bovine erythrocyte superoxide dismutase (SOD) has been determined at pH 7.0, with potassium ferricyanide as the mediator. The following thermodynamic parameters have been obtained (T = 25°C): E°' = 403±5 mV vs. NHE, ΔG°' = -9.31 kcal/mol, ΔH°' = -21.4 kcal/mol, ΔS°' = -40.7 eu, ΔS°'_(rc) = -25.1 eu. It is apparent from these results that ΔH°', rather than ΔS°', is the dominant factor in establishing the high redox potential of SOD. The large negative enthalpy of reduction may also reflect the factors which give SOD its high specificity toward reduction and oxidation by superoxide.

Expression of eph-family receptor tyrosine kinases and ephrins in the tadpole of the frog Xenopus laevis, and possible roles in the development of retinotectal topography

Assembling a nervous system requires exquisite specificity in the construction of neuronal connectivity. One method by which such specificity is implemented is the presence of chemical cues within the tissues, differentiating one region from another, and the presence of receptors for those cues on the surface of neurons and their axons that are navigating within this cellular environment.

Connections from one part of the nervous system to another often take the form of a topographic mapping. One widely studied model system that involves such a mapping is the vertebrate retinotectal projection-the set of connections between the eye and the optic tectum of the midbrain, which is the primary visual center in non-mammals and is homologous to the superior colliculus in mammals. In this projection the two-dimensional surface of the retina is mapped smoothly onto the two-dimensional surface of the tectum, such that light from neighboring points in visual space excites neighboring cells in the brain. This mapping is implemented at least in part via differential chemical cues in different regions of the tectum.

The Eph family of receptor tyrosine kinases and their cell-surface ligands, the ephrins, have been implicated in a wide variety of processes, generally involving cellular movement in response to extracellular cues. In particular, they possess expression patterns-i.e., complementary gradients of receptor in retina and ligand in tectum- and in vitro and in vivo activities and phenotypes-i.e., repulsive guidance of axons and defective mapping in mutants, respectively-consistent with the long-sought retinotectal chemical mapping cues.

The tadpole of Xenopus laevis, the South African clawed frog, is advantageous for in vivo retinotectal studies because of its transparency and manipulability. However, neither the expression patterns nor the retinotectal roles of these proteins have been well characterized in this system. We report here comprehensive descriptions in swimming stage tadpoles of the messenger RNA expression patterns of eleven known Xenopus Eph and ephrin genes, including xephrin-A3, which is novel, and xEphB2, whose expression pattern has not previously been published in detail. We also report the results of in vivo protein injection perturbation studies on Xenopus retinotectal topography, which were negative, and of in vitro axonal guidance assays, which suggest a previously unrecognized attractive activity of ephrins at low concentrations on retinal ganglion cell axons. This raises the possibility that these axons find their correct targets in part by seeking out a preferred concentration of ligands appropriate to their individual receptor expression levels, rather than by being repelled to greater or lesser degrees by the ephrins but attracted by some as-yet-unknown cue(s).

Physical characterization of the rack effect and hydrogen bond networks in blue copper proteins

A summary of previous research is presented that indicates that the purpose of a blue copper protein's fold and hydrogen bond network, aka, the rack effect, enforce a copper(II) geometry around the copper(I) ion in the metal site. In several blue copper proteins, the C-terminal histidine ligand becomes protonated and detaches from the copper in the reduced forms. Mutants of amicyanin from Paracoccus denitrificans were made to alter the hydrogen bond network and quantify the rack effect by pK_a shifts.

The pK_a's of mutant amicyanins have been measured by pH-dependent electrochemistry. P94F and P94A mutations loosen the Northern loop, allowing the reduced copper to adopt a relaxed conformation: the ability to relax drives the reduction potentials up. The measured potentials are 265 (wild type), 380 (P94A), and 415 (P94F) mV vs. NHE. The measured pK_a's are 7.0 (wild type), 6.3 (P94A), and 5.0 (P94F). The additional hydrogen bond to the thiolate in the mutants is indicated by a red-shift in the blue copper absorption and an increase in the parallel hyperfine splitting in the EPR spectrum. This hydrogen bond is invoked as the cause for the increased stability of the C-terminal imidazole.

Melting curves give a measure of the thermal stability of the protein. A thermodynamic intermediate with pH-dependent reversibility is revealed. Comparisons with the electrochemistry and apoamicyanin suggest that the intermediate involves the region of the protein near the metal site. This region is destabilized in the P94F mutant; coupled with the evidence that the imidazole is stabilized under the same conditions confirms an original concept of the rack effect: a high energy configuration is stabilized at a cost to the rest of the protein.

The Get3 ATPase drives unidirectional targeting of tail-anchored membrane proteins

Efficient and accurate localization of membrane proteins is essential to all cells and requires a complex cascade of interactions between protein machineries. This is exemplified in the recently discovered Guided Entry of Tail-anchored protein pathway, in which the central targeting factor Get3 must sequentially interact with three distinct binding partners (Get4, Get1 and Get2) to ensure the targeted delivery of Tail-anchored proteins to the endoplasmic reticulum membrane. To understand the molecular and energetic principles that provide the vectorial driving force of these interactions, we used a quantitative fluorescence approach combined with mechanistic enzymology to monitor the effector interactions of Get3 at each stage of Tail-anchored protein targeting. We show that nucleotide and membrane protein substrate generate a gradient of interaction energies that drive the cyclic and ordered transit of Get3 from Get4 to Get2 and lastly to Get1. These data also define how the Get3/Tail-anchored complex is captured, handed over, and disassembled by the Get1/2 receptor at the membrane, and reveal a novel role for Get4/5 in recycling Get3 from the endoplasmic reticulum membrane at the end of the targeting reaction. These results provide general insights into how complex cascades of protein interactions are coordinated and coupled to energy inputs in biological systems.

Biological electron transfer in copper proteins

Nature has used a variety of protein systems to mediate electron transfer. In this thesis I examine aspects of the control of biological electron transfer by two copper proteins that act as natural electron carriers.

In the first study, I have made a mutation to one of the ligand residues in the azurin blue copper center, methionine 121 changed to a glutamic acid. Studies of intramolecular electron transfer rates from that mutated center to covalently attached ruthenium complexes indicate that the weak axial methionine ligand is important not only for tuning the reduction potential of the blue copper site but also for maintaining the low reorganization energy that is important for fast electron transfer at long distances.

In the second study, I begin to examine the reorganization energy of the purple copper center in the CuA domain of subunit II of cytochrome c oxidase. In this copper center, the unpaired electron is delocalized over the entire binuclear site. Because long-range electron transfer into and out of this center occurs over long distances with very small driving forces, the reorganization energy of the CuA center has been predicted to be extremely low. I describe a strategy for measuring this reorganization energy starting with the construction of a series of mutations introducing surface histidines. These histidines can then be labeled with a series of ruthenium compounds that differ primarily in their reduction potentials. The electron transfer rates to these ruthenium compounds can then be used to determine the reorganization energy of the CuA site.

Computational design of self-assembling proteins and protein-DNA nanowires

Computational protein design (CPD) is a burgeoning field that uses a physical-chemical or knowledge-based scoring function to create protein variants with new or improved properties. This exciting approach has recently been used to generate proteins with entirely new functions, ones that are not observed in naturally occurring proteins. For example, several enzymes were designed to catalyze reactions that are not in the repertoire of any known natural enzyme. In these designs, novel catalytic activity was built de novo (from scratch) into a previously inert protein scaffold. In addition to de novo enzyme design, the computational design of protein-protein interactions can also be used to create novel functionality, such as neutralization of influenza. Our goal here was to design a protein that can self-assemble with DNA into nanowires. We used computational tools to homodimerize a transcription factor that binds a specific sequence of double-stranded DNA. We arranged the protein-protein and protein-DNA binding sites so that the self-assembly could occur in a linear fashion to generate nanowires. Upon mixing our designed protein homodimer with the double-stranded DNA, the molecules immediately self-assembled into nanowires. This nanowire topology was confirmed using atomic force microscopy. Co-crystal structure showed that the nanowire is assembled via the desired interactions. To the best of our knowledge, this is the first example of a protein-DNA self-assembly that does not rely on covalent interactions. We anticipate that this new material will stimulate further interest in the development of advanced biomaterials.