A de novo protein catalyzes the synthesis of semiconductor quantum dots.

De novo proteins constructed from novel amino acid sequences are distinct from proteins that evolved in nature. Construct K (ConK) is a binary-patterned de novo designed protein that rescues Escherichia coli from otherwise toxic concentrations of copper. ConK was recently found to bind the cofactor PLP (pyridoxal phosphate, the active form of vitamin B6). Here, we show that ConK catalyzes the desulfurization of cysteine to H2S, which can be used to synthesize CdS nanocrystals in solution. The CdS nanocrystals are approximately 3 nm, as measured by transmission electron microscope, with optical properties similar to those seen in chemically synthesized quantum dots. The CdS nanocrystals synthesized using ConK have slower growth rates and a different growth mechanism than those synthesized using natural biomineralization pathways. The slower growth rate yields CdS nanocrystals with two desirable properties not observed during biomineralization using natural proteins. First, CdS nanocrystals are predominantly of the zinc blende crystal phase; this is in stark contrast to natural biomineralization routes that produce a mixture of zinc blende and wurtzite phase CdS. Second, in contrast to the growth and eventual precipitation observed in natural biomineralization systems, the CdS nanocrystals produced by ConK stabilize at a final size. Future optimization of CdS nanocrystal growth using ConK-or other de novo proteins-may help to overcome the limits on nanocrystal quality typically observed from natural biomineralization by enabling the synthesis of more stable, high-quality quantum dots at room temperature.

A Completely De Novo ATPase from Combinatorial Protein Design.

Our understanding of biological chemistry is shaped by the observation that all life comes from other life-as Pasteur put it, omne vivum ex vivo. A key step in expanding our biochemical vocabulary is to recapitulate biogenic catalysis using non-natural sequences that did not arise from common ancestry. Here we describe an enzyme designed completely de novo that hydrolyzes ATP. This protein was designed to lack ß-sheet structure and is competitively inhibited by magnesium, two traits that are unlike natural ATPases.

A novel inhibitor of amyloid ß (Aß) peptide aggregation: from high throughput screening to efficacy in an animal model of Alzheimer disease.

Compelling evidence indicates that aggregation of the amyloid ß (Aß) peptide is a major underlying molecular culprit in Alzheimer disease. Specifically, soluble oligomers of the 42-residue peptide (Aß42) lead to a series of events that cause cellular dysfunction and neuronal death. Therefore, inhibiting Aß42 aggregation may be an effective strategy for the prevention and/or treatment of disease. We describe the implementation of a high throughput screen for inhibitors of Aß42 aggregation on a collection of 65,000 small molecules. Among several novel inhibitors isolated by the screen, compound D737 was most effective in inhibiting Aß42 aggregation and reducing Aß42-induced toxicity in cell culture. The protective activity of D737 was most significant in reducing the toxicity of high molecular weight oligomers of Aß42. The ability of D737 to prevent Aß42 aggregation protects against cellular dysfunction and reduces the production/accumulation of reactive oxygen species. Most importantly, treatment with D737 increases the life span and locomotive ability of flies in a Drosophila melanogaster model of Alzheimer disease.

Mutations that replace aromatic side chains promote aggregation of the Alzheimer's Aß peptide.

The aggregation of polypeptides into amyloid fibrils is associated with a number of human diseases. Because these fibrils--or intermediates on the aggregation pathway--play important roles in the etiology of disease, considerable effort has been expended to understand which features of the amino acid sequence promote aggregation. One feature suspected to direct aggregation is the π-stacking of aromatic residues. Such π-stacking interactions have also been proposed as the targets for various aromatic compounds that are known to inhibit aggregation. In the case of Alzheimer's disease, the aromatic side chains Phe19 and Phe20 in the wild-type amyloid beta (Aß) peptide have been implicated. To explicitly test whether the aromaticity of these side chains plays a role in aggregation, we replaced these two phenylalanine side chains with leucines or isoleucines. These residues have similar sizes and hydrophobicities as Phe but are not capable of π-stacking. Thioflavin-T fluorescence and electron microscopy demonstrate that replacement of residues 19 and 20 by Leu or Ile did not prevent aggregation, but rather enhanced amyloid formation. Further experiments showed that aromatic inhibitors of aggregation are as effective against Ile- and Leu-substituted versions of Aß42 as they are against wild-type Aß. These results suggest that aromatic π-stacking interactions are not critical for Aß aggregation or for the inhibition of Aß aggregation.

Small molecule microarrays enable the discovery of compounds that bind the Alzheimer's Aß peptide and reduce its cytotoxicity.

The amyloid-ß (Aß) aggregation pathway is a key target in efforts to discover therapeutics that prevent or delay the onset of Alzheimer's disease. Efforts at rational drug design, however, are hampered by uncertainties about the precise nature of the toxic aggregate. In contrast, high-throughput screening of compound libraries does not require a detailed understanding of the structure of the toxic species, and can provide an unbiased method for the discovery of small molecules that may lead to effective therapeutics. Here, we show that small molecule microarrays (SMMs) represent a particularly promising tool for identifying compounds that bind the Aß peptide. Microarray slides with thousands of compounds immobilized on their surface were screened for binding to fluorescently labeled Aß. Seventy-nine compounds were identified by the SMM screen, and then assayed for their ability to inhibit the Aß-induced killing of PC12 cells. Further experiments focused on exploring the mechanism of rescue for one of these compounds: Electron microscopy and Congo red binding showed that the compound enhances fibril formation, and suggest that it may rescue cells by accelerating Aß aggregation past an early toxic oligomer. These findings demonstrate that the SMM screen for binding to Aß is effective at identifying compounds that reduce Aß toxicity, and can reveal potential therapeutic leads without the biases inherent in methods that focus on inhibitors of aggregation.

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

The design of large libraries of well-folded de novo proteins is a powerful approach toward the ultimate goal of producing proteins with novel structures and functions for use in industry or medicine. A method for library design that incorporates both rational design and combinatorial diversity relies on the "binary patterning" of polar and nonpolar amino acids. Binary patterning is based on the premise that the appropriate arrangement of polar and nonpolar residues can direct a polypeptide chain to fold into amphipathic elements of secondary structure that anneal together to form a desired tertiary structure. A designed binary pattern exploits the periodicities inherent in protein secondary structure, and allows the identity of the side chain at each polar and nonpolar position to be varied combinatorially. This chapter provides an overview of the considerations necessary to use binary patterning to design libraries of novel proteins.

De novo proteins from binary-patterned combinatorial libraries.

Combinatorial libraries of well-folded de novo proteins can provide a rich source of reagents for the isolation of novel molecules for biotechnology and medicine. To produce libraries containing an abundance of well-folded sequences, we have developed a method that incorporates both rational design and combinatorial diversity. Our method specifies the "binary patterning" of polar and nonpolar amino acids, but allows combinatorial diversity of amino acid side chains at each polar and nonpolar site in the sequence. Protein design by binary patterning is based on the premise that the appropriate arrangement of polar and nonpolar residues can direct a polypeptide chain to fold into amphipathic elements of secondary structures, which anneal together to form a desired tertiary structure. A designed binary pattern exploits the periodicities inherent in protein secondary structure, while allowing the identity of the side chain at each polar and non-polar position to be varied combinatorially. This chapter provides an overview of the considerations necessary to design binary patterned libraries of novel proteins.

Nanografting de novo proteins onto gold surfaces.

The immobilization of novel proteins onto addressable locations on a flat surface has potential applications in a range of biotechnologies. Here we describe the nanopatterning of a de novo protein onto a gold surface. Patterning was achieved using a technique called nanografting, in which the tip of an atomic force microscope is used to disrupt a preexisting monolayer of alkanethiol molecules on a gold surface, thereby facilitating exchange with alternative thiol-linked molecules from the surrounding solution. The protein used for these studies was chosen from a designed combinatorial library of de novo sequences expressed in E. coli and was engineered to have a glycine-glycine-cysteine tag at its C-terminus, thereby enabling attachment to the gold surface through a single cysteine thiol. The average height of the grafted protein patterns was found to be somewhat higher than expected from the known NMR structure of the protein. Compression of the nanografted patches by an external force (below 10 nN) was reversible but showed some hysteresis. Interestingly, both the energy required to deform the immobilized protein patterns and the energy defined by the hysteresis loop were found to be of the same order as the energy required to unfold the monomeric protein in solution. These studies demonstrate the possibility of preparing nanometer scale protein arrays, lowering significantly the volume requirements of the protein samples necessary to fabricate protein-based biosensor arrays and thereby providing a base for increasing their sensitivity.

Sequence determinants of enhanced amyloidogenicity of Alzheimer A{beta}42 peptide relative to A{beta}40.

Aggregation of proteins into insoluble deposits is associated with a variety of human diseases. In Alzheimer disease, the aggregation of amyloid beta (Abeta) peptides is believed to play a key role in pathogenesis. Although the 40-mer (Abeta40) is produced in vivo at higher levels than the 42-mer (Abeta42), senile plaque in diseased brains is composed primarily of Abeta42. Likewise, in vitro, Abeta42 forms fibrils more rapidly than Abeta40. The enhanced amyloidogenicity of Abeta42 could be due simply to its greater length. Alternatively, specific properties of residues Ile(41) and Ala(42) might favor aggregation. To distinguish between these two possibilities, we constructed a library of sequences in which residues 41 and 42 were randomized. The aggregation behavior of the resulting sequences was assessed using a high throughput screen, based on the finding that fusions of Abeta42 to green fluorescence protein (GFP) prevent the folding and fluorescence of GFP, whereas mutations in Abeta42 that disrupt aggregation produce green fluorescent fusions. Correlations between the sequences of Abeta42 mutants and the fluorescence of Abeta42-GFP fusions in vivo were confirmed in vitro through biophysical studies of synthetic 42-residue peptides. The data reveal a strong correlation between aggregation propensity and the hydrophobicity and beta-sheet propensities of residues at positions 41 and 42. Moreover, several mutants containing hydrophilic residues and/or beta-sheet breakers at positions 41 and/or 42 were less prone to aggregate than Abeta40 wherein these two residues are deleted entirely. Thus, properties of the side chains at positions 41 and 42, rather than length per se, cause Abeta42 to aggregate more readily than Abeta40.

An intein-based genetic selection allows the construction of a high-quality library of binary patterned de novo protein sequences.

Combinatorial libraries of synthetic DNA are increasingly being used to identify and evolve proteins with novel folds and functions. An effective strategy for maximizing the diversity of these libraries relies on the assembly of large genes from smaller fragments of synthetic DNA. To optimize library assembly and screening, it is desirable to remove from the synthetic libraries any sequences that contain unintended frameshifts or stop codons. Although genetic selection systems can be used to accomplish this task, the tendency of individual segments to yield misfolded or aggregated products can decrease the effectiveness of these selections. Furthermore, individual protein domains may misfold when removed from their native context. We report the development and characterization of an in vivo system to preselect sequences that encode uninterrupted gene segments regardless of the foldedness of the encoded polypeptide. In this system, the inserted synthetic gene segment is separated from an intein/thymidylate synthase (TS) reporter domain by a polyasparagine linker, thereby permitting the TS reporter to fold and function independently of the folding and function of the segment-encoded polypeptide. TS-deficient Escherichia coli host cells survive on selective medium only if the insert is uninterrupted and in-frame, thereby allowing selection and amplification of desired sequences. We demonstrate that this system can be used as a highly effective preselection tool for the production of large, diverse and high-quality libraries of de novo protein sequences.

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.

Mutations that reduce aggregation of the Alzheimer's Abeta42 peptide: an unbiased search for the sequence determinants of Abeta amyloidogenesis.

The primary component of amyloid plaque in the brains of Alzheimer's patients is the 42 residue amyloid-beta-peptide (Abeta42). Although the amino acid residue sequence of Abeta42 is known, the molecular determinants of Abeta amyloidogenesis have not been elucidated. To facilitate an unbiased search for the sequence determinants of Abeta aggregation, we developed a genetic screen that couples a readily observable phenotype in E. coli to the ability of a mutation in Abeta42 to reduce aggregation. The screen is based on our finding that fusions of the wild-type Abeta42 sequence to green fluorescent protein (GFP) form insoluble aggregates in which GFP is inactive. Cells expressing such fusions do not fluoresce. To isolate variants of Abeta42 with reduced tendencies to aggregate, we constructed and screened libraries of Abeta42-GFP fusions in which the sequence of Abeta42 was mutated randomly. Cells expressing GFP fusions to soluble (non-aggregating) variants of Abeta42 exhibit green fluorescence. Implementation of this screen enabled the isolation of 36 variants of Abeta42 with reduced tendencies to aggregate. The sequences of most of these variants are consistent with previous models implicating hydrophobic regions as determinants of Abeta42 aggregation. Some of the variants, however, contain amino acid substitutions not implicated in pre-existing models of Abeta amyloidogenesis.