Binary Patterning Research Articles

Sequence periodicity and secondary structure propensity in model proteins

We explore the question of whether local effects (originating from the amino acids intrinsic secondary structure propensities) or nonlocal effects (reflecting the sequence of amino acids as a whole) play a larger role in determining the fold of globular proteins. Earlier circular dichroism studies have shown that the pattern of polar, non polar amino acids (nonlocal effect) dominates over the amino acid intrinsic propensity (local effect) in determining the secondary structure of oligomeric peptides. In this article, we present a coarse grained computational model that allows us to quantitatively estimate the role of local and nonlocal factors in determining both the secondary and tertiary structure of small, globular proteins. The amino acid intrinsic secondary structure propensity is modeled by a dihedral potential term. This dihedral potential is parametrized to match with experimental measurements of secondary structure propensity. Similarly, the magnitude of the attraction between hydrophobic residues is parametrized to match the experimental transfer free energies of hydrophobic amino acids. Under these parametrization conditions, we systematically explore the degree of frustration a given polar, non polar pattern can tolerate when the secondary structure intrinsic propensities are in opposition to it. When the parameters are in the biophysically relevant range, we observe that the fold of small, globular proteins is determined by the pattern of polar, non polar amino acids regardless of their instrinsic secondary structure propensities. Our simulations shed new light on previous observations that tertiary interactions are more influential in determining protein structure than secondary structure propensity. The fact that this can be inferred using a simple polymer model that lacks most of the biochemical details points to the fundamental importance of binary patterning in governing folding.

Cofactor binding and enzymatic activity in an unevolved superfamily of de novo designed 4-helix bundle proteins.

To probe the potential for enzymatic activity in unevolved amino acid sequence space, we created a combinatorial library of de novo 4-helix bundle proteins. This collection of novel proteins can be considered an "artificial superfamily" of helical bundles. The superfamily of 102-residue proteins was designed using binary patterning of polar and nonpolar residues, and expressed in Escherichia coli from a library of synthetic genes. Sequences from the library were screened for a range of biological functions including heme binding and peroxidase, esterase, and lipase activities. Proteins exhibiting these functions were purified and characterized biochemically. The majority of de novo proteins from this superfamily bound the heme cofactor, and a sizable fraction of the proteins showed activity significantly above background for at least one of the tested enzymatic activities. Moreover, several of the designed 4-helix bundles proteins showed activity in all of the assays, thereby demonstrating the functional promiscuity of unevolved proteins. These studies reveal that de novo proteins-which have neither been designed for function, nor subjected to evolutionary pressure (either in vivo or in vitro)-can provide rudimentary activities and serve as a "feedstock" for evolution.

Binary and Gray-Scale Patterning of Chemical Functionality on Polymer Films

We present a facile technique for the gray-scale chemical functionalization of polymer surfaces with high dynamic range. We demonstrate the use of this technique to create amine-functionalized substrates that are used for the patterned binding of fluorophores and the patterned synthesis of peptides. Studies of the behavior of the model organism Dictyostelium discoideum indicate the biocompatibility of the functionalized substrates.

Integrated Direct DNA/Protein Patterning and Microfabrication by Focused Ion Beam Milling

Single and binary component patterning and integrated microfabrication of biomolecules, such as DNA and proteins, can be achieved by focused ion-beam (FIB) biolithography. Well-defined micropatterns are obtained by FIB milling on biomolecules immobilized on SiO2 wafers and protected by a thin Au film. The retention of biofunctionality is excellent (68–90%) and a feature size of down to 500 nm can be achieved for the patterns without significant loss of functionality.

Structure and dynamics of de novo proteins from a designed superfamily of 4‐helix bundles

Libraries of de novo proteins provide an opportunity to explore the structural and functional potential of biological molecules that have not been biased by billions of years of evolutionary selection. Given the enormity of sequence space, a rational approach to library design is likely to yield a higher fraction of folded and functional proteins than a stochastic sampling of random sequences. We previously investigated the potential of library design by binary patterning of hydrophobic and hydrophilic amino acids. The structure of the most stable protein from a binary patterned library of de novo 4-helix bundles was solved previously and shown to be consistent with the design. One structure, however, cannot fully assess the potential of the design strategy, nor can it account for differences in the stabilities of individual proteins. To more fully probe the quality of the library, we now report the NMR structure of a second protein, S-836. Protein S-836 proved to be a 4-helix bundle, consistent with design. The similarity between the two solved structures reinforces previous evidence that binary patterning can encode stable, 4-helix bundles. Despite their global similarities, the two proteins have cores that are packed at different degrees of tightness. The relationship between packing and dynamics was probed using the Modelfree approach, which showed that regions containing a high frequency of chemical exchange coincide with less well-packed side chains. These studies show (1) that binary patterning can drive folding into a particular topology without the explicit design of residue-by-residue packing, and (2) that within a superfamily of binary patterned proteins, the structures and dynamics of individual proteins are modulated by the identity and packing of residues in the hydrophobic core.

Newly Synthesized Branch-type Aromatic Oxadiazole Polymer and Binary Fluorescence Patterning on its Film

Aromatic side-chain oxadiazole polymer linked with 9,9'-dioctylfluorene was successfully synthesized via Suzuki coupling reaction. Hydroxyphenyl group was attached in the 2-position of the oxadiazole unit in the polymer side chain to control the optical properties of the polymer. We confirmed the presence of the t-butoxycarbonyl group on the hydroxyl group using thermogravimetric analysis, which was incorporated to avoid side reaction during polymerization. We also performed the simple and easy fabrication method for the dual fluorescence image using photochemical cleavage of the t-butoxycarbonyl group from the polymer to induce fluorescence color changes before and after UV irradiation.

Electrochemical and ligand binding studies of a de novo heme protein

Heme proteins can perform a variety of electrochemical functions. While natural heme proteins carry out particular functions selected by biological evolution, artificial heme proteins, in principle, can be tailored to suit specified technological applications. Here we describe initial characterization of the electrochemical properties of a de novo heme protein, S824C. Protein S824C is a four-helix bundle derived from a library of sequences that was designed by binary patterning of polar and nonpolar amino acids. Protein S824C was immobilized on a gold electrode and the formal potential of heme–protein complex was studied as a function of pH and ionic strength. The binding of exogenous N-donor ligands to heme/S824C was monitored by measuring shifts in the potential that occurred upon addition of various concentrations of imidazole or pyridine derivatives. The response of heme/S824C to these ligands was then compared to the response of isolated heme (without protein) to the same ligands. The observed shifts in potential depended on both the concentration and the structure of the added ligand. Small changes in structure of the ligand (e.g. pyridine versus 2-amino pyridine) produced significant shifts in the potential of the heme–protein. The observed shifts correlate to the differential binding of the N-donor molecules to the oxidized and reduced states of the heme. Further, it was observed that the electrochemical response of the buried heme in heme/S824C differed significantly from that of isolated heme. These studies demonstrate that the structure of the de novo protein modulates the binding of N-donor ligands to heme.

Enzyme-like proteins from an unselected library of designed amino acid sequences.

Combinatorial libraries of de novo amino acid sequences can provide a rich source of diversity for the discovery of novel proteins with interesting and important activities. However, since arbitrary sequences rarely fold into well ordered protein-like structures, randomly generated libraries will yield functional proteins only very rarely. To enhance the likelihood of finding functional de novo proteins, we use binary patterning of polar and non-polar amino acids to design focused libraries of sequences that are predisposed to fold into ordered structures. Proteins isolated from binary patterned libraries have been shown previously to fold into well ordered and native-like three-dimensional structures. To probe the potential of such libraries to also yield proteins with enzyme-like activity, we measured the esterase activity of S-824, a de novo binary patterned protein whose alpha-helical three-dimensional structure was reported recently. Protein S-824 displayed a rate enhancement (k(cat)/k(uncat)) of 8700. The observed activity is similar to, or better than, that observed for several esterases designed previously using rational design or automated computational methods. Moreover, the observed activity rivals those of the first catalytic antibodies. To assess whether the activity of S-824 is representative of other proteins in binary patterned libraries, we measured the esterase activity of six additional proteins from two libraries. These libraries were 'naïve' in that they were neither designed to bind substrate, nor subjected to high throughput screens for activity. All six of the additional proteins displayed esterase activity significantly above background. These findings demonstrate that novel proteins with enzyme-like properties are surprisingly common in focused libraries designed by binary patterning. Moreover, the activity of these unselected proteins provides a reference state for the levels of activity that have been obtained by selection and/or computational design.

Solution structure of a de novo protein from a designed combinatorial library.

Combinatorial libraries of de novo amino acid sequences can provide a rich source of diversity for the discovery of novel proteins. Randomly generated sequences, however, rarely fold into well ordered protein-like structures. To enhance the quality of a library, diversity must be focused into those regions of sequence space most likely to yield well folded structures. We have constructed focused libraries of de novo sequences by designing the binary pattern of polar and nonpolar amino acids to favor structures that contain abundant secondary structure, while simultaneously burying hydrophobic side chains in the protein interior and exposing hydrophilic side chains to solvent. Because binary patterning specifies only the polar/nonpolar periodicity, but not the identities of the side chains, detailed structural features, including packing interactions, cannot be designed a priori. Can binary patterned libraries nonetheless encode well folded proteins? An unambiguous answer to this question requires determination of a 3D structure. We used NMR spectroscopy to determine the structure of S-824, a novel protein from a recently constructed library of 102-residue sequences. This library is "naïve" in that it has not been subjected to high-throughput screens or directed evolution. The experimentally determined structure of S-824 is a four-helix bundle, as specified by the design. As dictated by the binary-code strategy, nonpolar side chains are buried in the protein interior, and polar side chains are exposed to solvent. The polypeptide backbone and buried side chains are well ordered, demonstrating that S-824 is not a molten globule and forms a unique structure. These results show that amino acid sequences that have neither been selected by evolution, nor designed by computer, nor isolated by high-throughput screening, can form native-like structures. These findings validate the binary-code strategy as an effective method for producing vast collections of well folded de novo proteins.

Stably folded de novo proteins from a designed combinatorial library.

Binary patterning of polar and nonpolar amino acids has been used as the key design feature for constructing large combinatorial libraries of de novo proteins. Each position in a binary patterned sequence is designed explicitly to be either polar or nonpolar; however, the precise identities of these amino acids are varied extensively. The combinatorial underpinnings of the "binary code" strategy preclude explicit design of particular side chains at specified positions. Therefore, packing interactions cannot be specified a priori. To assess whether the binary code strategy can nonetheless produce well-folded de novo proteins, we constructed a second-generation library based upon a new structural scaffold designed to fold into 102-residue four-helix bundles. Characterization of five proteins chosen arbitrarily from this new library revealed that (1) all are alpha-helical and quite stable; (2) four of the five contain an abundance of tertiary interactions indicative of well-ordered structures; and (3) one protein forms a well-folded structure with native-like features. The proteins from this new 102-residue library are substantially more stable and dramatically more native-like than those from an earlier binary patterned library of 74-residue sequences. These findings demonstrate that chain length is a crucial determinant of structural order in libraries of de novo four-helix bundles. Moreover, these results show that the binary code strategy--if applied to an appropriately designed structural scaffold--can generate large collections of stably folded and/or native-like proteins.

Construction and characterization of protein libraries composed of secondary structure modules.

Only a minute fraction of all possible protein sequences can exist in the genomes of all life forms. To explore whether physicochemical constraints or a lack of need causes the paucity of different protein folds, we set out to construct protein libraries without any restriction of topology. We generated different libraries (all alpha-helix, all beta-strand, and alpha-helix plus beta-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (alpha-helix, beta-strand, and beta-turn) in various proportions, based primarily on the patterning of polar and nonpolar residues. We wished to explore that part of sequence space that is rich in secondary structure. The analysis of randomly chosen clones from each of the libraries showed that, despite the low sequence homology to known protein sequences, a substantial proportion of the library members containing alpha-helix modules were indeed helical, possess a defined oligomerization state, and showed cooperative chemical unfolding behavior. On the other hand, proteins composed of mainly beta-strand modules tended to form amyloid-like fibrils and were among the least soluble proteins ever reported. We found that a large fraction of members in non-beta-strand-containing protein libraries that are distant from natural proteins in sequence space possess unexpectedly favorable properties. These results reinforce the efficacy of applying binary patterning to design proteins with native-like properties despite lack of restriction in topology. Because of the intrinsic tendency of beta-strand modules to aggregate, their presence requires precise topologic arrangement to prevent fibril formation.

Self-assembled monolayers from a designed combinatorial library of de novo beta-sheet proteins.

A variety of naturally occurring biomaterials owe their unusual structural and mechanical properties to layers of beta-sheet proteins laminated between layers of inorganic mineral. To explore the possibility of fabricating novel two-dimensional protein layers, we studied the self-assembly properties of de novo proteins from a designed combinatorial library. Each protein in the library has a distinct 63 amino acid sequence, yet they all share an identical binary pattern of polar and nonpolar residues, which was designed to favor the formation of six-stranded amphiphilic beta-sheets. Characterization of proteins isolated from the library demonstrates that (i) they self assemble into monolayers at an air/water interface; (ii) the monolayers are dominated by beta-sheet secondary structure, as shown by both circular dichroism and infrared spectroscopies; and (iii) the measured areas (500- 600 A(2)) of individual protein molecules in the monolayers match those expected for proteins folded into amphiphilic beta-sheets. The finding that similar structures are formed by distinctly different protein sequences suggests that assembly into beta-sheet monolayers can be encoded by binary patterning of polar and nonpolar amino acids. Moreover, because the designed binary pattern is compatible with a wide variety of different sequences, it may be possible to fabricate beta-sheet monolayers by using combinations of side chains that are explicitly designed to favor particular applications of novel biomaterials.

De novo design and characterization of an apolar helical hairpin peptide at atomic resolution: Compaction mediated by weak interactions.

Design of helical super secondary structural motifs is expected to provide important scaffolds to incorporate functional sites, thus allowing the engineering of novel miniproteins with function. An alpha,beta-dehydrophenylalanine containing 21-residue apolar peptide was designed to mimic the helical hairpin motif by using a simple geometrical design strategy. The synthetic peptide folds into the desired structure as assessed crystallographically at 1.0-A resolution. The two helices of the helical-hairpin motif, connected by a flexible (Gly)(4) linker, are docked to each other by the concerted influence of weak interactions. The folding of the peptide without binary patterning of amino acids, disulfide bonds, or metal ions is a remarkable observation. The results demonstrate that preferred interactions among the hydrophobic residues selectively discriminate their putative partners in space, leading to the unique folding of the peptide, also a hallmark of the unique folding of hydrophobic core in globular proteins. We demonstrate here the engineering of molecules by using weak interactions pointing to their possible further exploitation in the de novo design of protein super secondary structural elements.

Achieving stability and conformational specificity in designed proteins via binary patterning

We have developed a method to determine the optimal binary pattern (arrangement of hydrophobic and polar amino acids) of a target protein fold prior to amino acid sequence selection in protein design studies. A solvent accessible surface is generated for a target fold using its backbone coordinates and “generic” side-chains, which are constructs whose size and shape are similar to an average amino acid. Each position is classified as hydrophobic or polar according to the solvent exposure of its generic side-chain. The method was tested by analyzing a set of proteins in the Protein Data Bank and by experimentally constructing and analyzing a set of engrailed homeodomain variants whose binary patterns were systematically varied. Selection of the optimal binary pattern results in a designed protein that is monomeric, well-folded, and hyperthermophilic. Homeodomain variants with fewer hydrophobic residues are destabilized, while additional hydrophobic residues induce aggregation. Binary patterning, in conjunction with a force field that models folded state energies, appears sufficient to satisfy two basic goals of protein design: stability and conformational specificity.

Cooperative thermal denaturation of proteins designed by binary patterning of polar and nonpolar amino acids.

We previously reported a combinatorial strategy for designing alpha-helical proteins by assigning only the binary patterning of polar or nonpolar residues [Kamtekar, S., Schiffer, J. M., Xiong, H. Y., Babik, J. M., and Hecht, M. H. (1993) Science 262, 1680-1685]. Here we describe the finding that approximately half of the proteins in the original collection display some level of cooperativity in their thermal denaturation profiles. Many are monomeric in solution, demonstrating that the observed cooperativity is not merely a consequence of oligomerization. These findings demonstrate that although the combinatorial nature of the design strategy precludes explicit design of side-chain packing, binary patterning incorporates sufficient sequence information to generate de novo proteins with cooperatively folded structures. As binary partitioning of polar and nonpolar amino acids is an intrinsic part of the genetic code, these findings may bear on the early evolution of native proteins.

De novo amyloid proteins from designed combinatorial libraries.

Amyloid deposits are associated with several neurodegenerative diseases, including Alzheimer's disease and the prion diseases. The amyloid fibrils isolated from these different diseases share similar structural features. However, the protein sequences that assemble into these fibrils differ substantially from one disease to another. To probe the relationship between amino acid sequence and the propensity to form amyloid, we studied a combinatorial library of sequences designed de novo. All sequences in the library were designed to share an identical pattern of alternating polar and nonpolar residues, but the precise identities of these side chains were not constrained and were varied combinatorially. The resulting proteins self-assemble into large oligomers visible by electron microscopy as amyloid-like fibrils. Like natural amyloid, the de novo fibrils are composed of beta-sheet secondary structure and bind the diagnostic dye, Congo red. Thus, binary patterning of polar and nonpolar residues arranged in alternating periodicity can direct protein sequences to form fibrils resembling amyloid. The model amyloid fibrils assemble and disassemble reversibly, providing a tractable system for both basic studies into the mechanisms of fibril assembly and the development of molecular therapies that interfere with this assembly.

A Protein Designed by Binary Patterning of Polar and Nonpolar Amino Acids Displays Native-like Properties

Large collections of de novo α-helical proteins can be constructed by using combinatorial methods based on a “binary code” for protein design, in which the sequence locations of polar and nonpolar amino acids are specified explicitly, but the precise identities of these residues are varied extensively. We demonstrate that a 75-residue protein chosen from such a binary code collection displays several properties similar to those of native proteins: (i) Both the chemically induced and thermally induced denaturations are cooperative; (ii) addition of the hydrophobic dye 1-analinonaphthalene-8-sulfonate (ANS) yields only minimal fluorescence; (iii) the NMR spectrum shows significant chemical shift dispersion in both the amide and methyl regions; and (iv) amide protons are protected from exchange to an extent observed in some natural proteins. These results demonstrate that binary patterning of polar and nonpolar amino acids can serve as the basis for initial steps toward the design of novel proteins with nat...

ChemInform Abstract: Protein Design by Binary Patterning of Polar and Nonpolar Amino Acids.

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Protein design by binary patterning of polar and nonpolar amino acids.

A general strategy is described for the de novo design of proteins. In this strategy the sequence locations of hydrophobic and hydrophilic residues were specified explicitly, but the precise identities of the side chains were not constrained and varied extensively. This strategy was tested by constructing a large collection of synthetic genes whose protein products were designed to fold into four-helix bundle proteins. Each gene encoded a different amino acid sequence, but all sequences shared the same pattern of polar and nonpolar residues. Characterization of the expressed proteins indicated that most of the designed sequences folded into compact alpha-helical structures. Thus, a simple binary code of polar and nonpolar residues arranged in the appropriate order can drive polypeptide chains to collapse into globular alpha-helical folds.