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J R Beasley

Publications and source records attributed to J R Beasley.

8 recordsLinked to original sources
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

Partially formed native tertiary interactions in the A-state of cytochrome c.

Considerable insight into protein structure, stability, and folding has been obtained from studies of non-native states. We have studied the extent of native tertiary contacts in one such molecule, the A-state of yeast iso-1-ferricytochrome c. Previously, we showed that the interface between the N and C-terminal helices is completely formed in the A-state. Here, we focus on interactions essential for forming the heme pocket of eukaryotic cytochromes c. To determine the extent of these interactions, we used saturation mutagenesis at the evolutionarily invariant residue leucine 68, and measured the free energy of denaturation for the native states and the A-states of functional variants. We show that, unlike the interaction between the terminal helices, the native interactions between the 60s helix and the rest of the protein are not completely formed in the A-state.

Codon, Terminator↗
PubMed1998

Unexpected frameshifts from gene to expressed protein in a phage-displayed peptide library.

A library of long peptides displayed on the pIII protein of filamentous phage was used in biopanning experiments against several protein targets. We find that a large percentage of phage clones that bind specifically to a target contain peptide-encoding genes that do not have an ORF. Instead, the reading frame is either interrupted by one or more nonsuppressed stop codons, or a post-transcriptional frameshift is needed to account for the expression of the minor phage coat protein pIII. The percentage of frameshifted clones varies depending on the target. It can be as high as 90% for clones specific for soluble forms of certain cytokine receptors. Conversely, biopanning against four mAbs did not yield any frameshifted clones. Our studies focused on one clone that binds specifically to rat growth hormone binding protein (GHBP) yet does not have an ORF. A secondary peptide library containing random mutations of this sequence was constructed and panned against GHBP to optimize and correct the reading frame. In the last round (round two) of panning with this library, none of the phage clones that bound to GHBP had an ORF. However, careful analysis of these clones allowed us to design a synthetic peptide capable of binding to GHBP. The results of this study indicate that ORFs are not required to obtain gene expression of the minor coat protein of filamentous phage and suggest that some ORF- clones may have a selective advantage over the clones having ORFs.

Amino Acid Sequence↗
PubMed1996

Requirements for perpendicular helix pairing.

Cassette mutagenesis was used to produce a library of mutations at the interface of the N- and C-terminal helices of Saccharomyces cerevisiae iso-1-cytochrome c. The library is random and comprises > 98% mutations. Over 11,000 candidates were assayed for function by selecting for the ability of yeast, with the mutated gene as their sole cytochrome c source, to grow on nonfermentable carbon sources. We estimate that approximately 0.5% of the 160,000 total amino acid combinations at these four residues result in a functional cytochrome c. Significant correlations are found between the phenotype of yeast harboring the alleles and both the Dayhoff mutation matrix and transfer free energies (cyclohexane-to-water and n-octanol-to-water). Similar correlations are observed with respect to growth rate. Finally, sequences that are consistent with function follow a binary amino acid pattern.

Amino Acid Sequence↗
PubMed1995

Protein thermal denaturation, side-chain models, and evolution: amino acid substitutions at a conserved helix-helix interface.

Random mutant libraries with substitutions at the interface between the N- and C-terminal helices of Saccharomyces cerevisiae iso-1-cytochrome c were screened. All residue combinations that have been identified in naturally occurring cytochrome c sequences are found in the libraries. Mutants with these combinations are biologically functional. Enthalpies, heat capacities, and midpoint temperatures of denaturation are used to determine the entropy and Gibbs free energy of denaturation (delta GD) for the ferri form of the wild-type protein and 13 interface variants. Changes in delta GD cannot be allocated solely to enthalpic or entropic effects, but there is no evidence of enthalpy-entropy compensation. The lack of additivity of delta GD values for single versus multiple amino acid substitutions indicates that the helices interact thermodynamically. Changes in delta GD are not in accord with helix propensities, indicating that interactions between the helices and the rest of the protein outweigh helix propensity. Comparison of delta GD values for the interface variants and nearly 90 non-cytochrome c variants to side-chain model data leads to several conclusions. First, hydrocarbon side chains react to burial-like transfer from water to cyclohexane, but even weakly polar side chains respond differently. Second, despite octanol being a poor model for protein interiors, octanol-to-water transfer free energies are useful stability predictors for changing large hydrocarbon side chains to smaller ones. Third, unlike cyclohexane and octanol, the Dayhoff mutation matrix predicts stability changes for a variety of substitutions, even at interacting sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Design of a ruthenium-cytochrome c derivative to measure electron transfer to the initial acceptor in cytochrome c oxidase.

A ruthenium-labeled cytochrome c derivative was prepared to meet two design criteria: the ruthenium group must transfer an electron rapidly to the heme group, but not alter the interaction with cytochrome c oxidase. Site-directed mutagenesis was used to replace His39 on the backside of yeast C102T iso-1-cytochrome c with a cysteine residue, and the single sulfhydryl group was labeled with (4-bromomethyl-4' methylbipyridine) (bis-bipyridine)ruthenium(II) to form Ru-39-cytochrome c (cyt c). There is an efficient pathway for electron transfer from the ruthenium group to the heme group of Ru-39-cyt c comprising 13 covalent bonds and one hydrogen bond. Electron transfer from the excited state Ru(II*) to ferric heme c occurred with a rate constant of (6.0 +/- 2.0) x 10(5) s-1, followed by electron transfer from ferrous heme c to Ru(III) with a rate constant of (1.0 +/- 0.2) x 10(6) s-1. Laser excitation of a complex between Ru-39-cyt c and beef cytochrome c oxidase in low ionic strength buffer (5 mM phosphate, pH7) resulted in electron transfer from photoreduced heme c to CuA with a rate constant of (6 +/- 2) x 10(4) s-1, followed by electron transfer from CuA to heme a with a rate constant of (1.8 +/- 0.3) x 10(4) s-1. Increasing the ionic strength to 100 mM leads to bimolecular kinetics as the complex is dissociated. The second-order rate constant is (2.5 +/- 0.4) x 10(7) M-1s-1 at 230 mM ionic strength, nearly the same as that of wild-type iso-1-cytochrome c.

Animals↗