Biomedical subjects

Michael H Hecht

Publications and source records attributed to Michael H Hecht.

At least 19 recordsLinked to original sources

PubMed2007

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.

Amino Acid Sequence↗

PubMed2006

Generic hydrophobic residues are sufficient to promote aggregation of the Alzheimer's Abeta42 peptide.

One hundred years ago, Alois Alzheimer observed a relationship between cognitive impairment and the presence of plaque in the brains of patients suffering from the disease that bears his name. The plaque was subsequently shown to be composed primarily of a 42-residue peptide called amyloid beta (Abeta) 42. Despite the importance of Abeta42 aggregation in the molecular etiology of Alzheimer's disease, the amino acid sequence determinants of this process have yet to be elucidated. Although stretches of hydrophobic residues in the C-terminal half of Abeta42 have been implicated, the mechanism by which these residues promote aggregation remains unclear. In particular, it is not known whether the side chains of these hydrophobic residues mediate specific interactions that direct self-assembly or, alternatively, whether hydrophobicity per se at these positions is sufficient to promote aggregation. To distinguish between these two possibilities, we substituted 12 hydrophobic residues in the C-terminal half of Abeta42 with random nonpolar residues. The mutant sequences were screened by using a fusion of Abeta42 to GFP. Because aggregation of Abeta42 prevents folding of the GFP reporter, this screen readily distinguishes aggregating from nonaggregating variants of Abeta42. Application of the screen demonstrated that, despite the presence of 8-12 mutations, all of the sequences aggregated. To confirm these results, several of the mutant sequences were prepared as synthetic peptides and shown to form amyloid fibrils similar to those of WT Abeta42. These findings indicate that hydrophobic stretches in the sequence of Abeta42, rather than specific side chains, are sufficient to promote aggregation.

Alzheimer Disease↗

PubMed2006

A high-throughput screen for compounds that inhibit aggregation of the Alzheimer's peptide.

Aggregation of the Alzheimer's peptide Abeta produces toxic multimeric species that play a key role in the development of Alzheimer's disease. Compounds that inhibit this aggregation may prove useful as therapeutic agents for the prevention or treatment of Alzheimer's disease. Although aggregation inhibitors may already exist in combinatorial libraries, finding these compounds in a cost-effective high-throughput manner poses an enormous challenge. To meet this challenge, we have developed a novel high-throughput screen capable of isolating inhibitors of Abeta aggregation from large libraries of inactive candidates. The screen uses a fusion of Abeta42 to GFP. In the absence of inhibition, the rapid misfolding and aggregation of Abeta42 causes the entire fusion protein to misfold, thereby preventing fluorescence. Compounds that inhibit Abeta42 aggregation enable GFP to fold into its native structure and be identified by the resulting fluorescent signal. By implementing the screen on a pilot library of triazine derivatives, we have identified several putative inhibitors. One of the selected compounds was studied in detail by a series of biochemical and biophysical methods. These studies confirmed that the selected compound inhibits aggregation of synthetic Abeta42 peptide. The fluorescence-based method described here is rapid and inexpensive and can be used to screen large libraries for inhibitors of Abeta42 aggregation and/or amyloidogenesis.

Alzheimer Disease↗

PubMed2006

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.

Binding Sites↗

PubMed2006

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.

Peptide Library↗

PubMed2006

Combinatorial approaches to probe the sequence determinants of protein aggregation and amyloidogenicity.

Elucidation of the molecular determinants that drive proteins to aggregate is important both to advance our fundamental understanding of protein folding and misfolding, and as a step towards successful intervention in human disease. Combinatorial strategies enable unbiased and model-free approaches to probe sequence/structure relationships. Through the use of combinatorial methods, it is possible (i) to probe the sequence determinants of natural amyloid proteins by screening libraries of amino acid substitutions (mutations) to identify those that prevent amyloid formation; and (ii) to test new hypotheses about the mechanism of formation of amyloid fibrils by using these hypotheses to guide the design of combinatorial libraries of de novo amyloid-like proteins. Here, we review how these two approaches have been used to study the molecular determinants of protein aggregation and amyloidogenicity.

Amino Acid Sequence↗

PubMed2005

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.

Adsorption↗

PubMed2005

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.

Alzheimer Disease↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

Ab initio prediction of the three-dimensional structure of a de novo designed protein: a double-blind case study.

Ab initio structure prediction and de novo protein design are two problems at the forefront of research in the fields of structural biology and chemistry. The goal of ab initio structure prediction of proteins is to correctly characterize the 3D structure of a protein using only the amino acid sequence as input. De novo protein design involves the production of novel protein sequences that adopt a desired fold. In this work, the results of a double-blind study are presented in which a new ab initio method was successfully used to predict the 3D structure of a protein designed through an experimental approach using binary patterned combinatorial libraries of de novo sequences. The predicted structure, which was produced before the experimental structure was known and without consideration of the design goals, and the final NMR analysis both characterize this protein as a 4-helix bundle. The similarity of these structures is evidenced by both small RMSD values between the coordinates of the two structures and a detailed analysis of the helical packing.

Algorithms↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2004

De novo proteins from designed combinatorial libraries.

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. Randomly generated sequences, however, rarely fold into well-ordered proteinlike structures. To enhance the quality of a library, features of rational design must be used to focus sequence diversity into those regions of sequence space that are most likely to yield folded structures. This review describes how focused libraries can be constructed by designing the binary pattern of polar and nonpolar amino acids to favor proteins that contain abundant secondary structure, while simultaneously burying hydrophobic side chains and exposing hydrophilic side chains to solvent. The "binary code" for protein design was used to construct several libraries of de novo proteins, including both alpha-helical and beta-sheet structures. The recently determined solution structure of a binary patterned four-helix bundle is well ordered, thereby demonstrating that sequences that have neither been selected by evolution (in vivo or in vitro) nor designed by computer can form nativelike proteins. Examples are presented demonstrating how binary patterned libraries have successfully produced well-ordered structures, cofactor binding, catalytic activity, self-assembled monolayers, amyloid-like nanofibrils, and protein-based biomaterials.

Models, Chemical↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2003

Mars Surveyor Program '01 Mars Environmental Compatibility Assessment wet chemistry lab: a sensor array for chemical analysis of the Martian soil.

The Mars Environmental Compatibility Assessment (MECA) instrument was designed, built, and flight qualified for the now canceled MSP (Mars Surveyor Program) '01 Lander. The MECA package consisted of a microscope, electrometer, material patch plates, and a wet chemistry laboratory (WCL). The primary goal of MECA was to analyze the Martian soil (regolith) for possible hazards to future astronauts and to provide a better understanding of Martian regolith geochemistry. The purpose of the WCL was to analyze for a range of soluble ionic chemical species and electrochemical parameters. The heart of the WCL was a sensor array of electrochemically based ion-selective electrodes (ISE). After 20 months storage at -23 degrees C and subsequent extended freeze/thawing cycles, WCL sensors were evaluated to determine both their physical durability and analytical responses. A fractional factorial calibration of the sensors was used to obtain slope, intercept, and all necessary selectivity coefficients simultaneously for selected ISEs. This calibration was used to model five cation and three anion sensors. These data were subsequently used to determine concentrations of several ions in two soil leachate simulants (based on terrestrial seawater and hypothesized Mars brine) and four actual soil samples. The WCL results were compared to simulant and soil samples using ion chromatography and inductively coupled plasma optical emission spectroscopy. The results showed that flight qualification and prolonged low-temperature storage conditions had minimal effects on the sensors. In addition, the analytical optimization method provided quantitative and qualitative data that could be used to accurately identify the chemical composition of the simulants and soils. The WCL has the ability to provide data that can be used to "read" the chemical, geological, and climatic history of Mars, as well as the potential habitability of its regolith.

Anions↗

PubMed2003

Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries.

We previously reported the de novo design of combinatorial libraries of proteins targeted to fold into four-helix bundles. The sequences of these proteins were designed using a binary code strategy in which each position in the linear sequence is designated as either polar or nonpolar, but the exact identity of the amino acid at each position is varied combinatorially. We subsequently reported that approximately half of these binary coded proteins were capable of binding heme. These de novo heme-binding proteins showed CO binding characteristics similar to natural heme proteins, and several were active as peroxidases. Here we analyze the midpoint reduction potentials and heme binding stoichiometries of several of these de novo heme proteins. All the proteins bound heme with a 1:1 stoichiometry. The reduction potentials ranged from -112 to -176 mV. We suggest that this represents an estimate of the default range of potentials for heme proteins that have neither been prejudiced by rational design nor selected by evolution.

Animals↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2002

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.

Alzheimer Disease↗

PubMed2002

Template-directed assembly of a de novo designed protein.

Many naturally occurring biomaterials are composed of laminated structures in which layers of beta-sheet proteins alternate with layers of inorganic mineral. These ordered laminates often have structural and mechanical properties that differ significantly from those of nonbiological materials. An important step in the construction of novel biomaterials is the creation of composites wherein a de novo designed protein assembles into an ordered structure. To achieve this goal, we layered a de novo protein onto the surface of highly ordered pyrolytic graphite (HOPG). The protein was derived from a combinatorial library of novel sequences designed to fold into amphiphilic beta-sheet structures. Atomic force microscopy reveals that the protein assembles on the HOPG surface into ordered fibers aligned in three orientations at 120 degrees to each other. The symmetry and extent of the ordered regions indicate that the hexagonal lattice underlying the graphite surface templates assembly of millions of protein molecules into a highly ordered structure.

Amino Acid Sequence↗