Biomedical subjects

Scott C Meyer

Publications and source records attributed to Scott C Meyer.

4 recordsLinked to original sources

PubMed2006

Inhibition of beta-amyloid fibrillization by directed evolution of a beta-sheet presenting miniature protein.

We describe the directed evolution of a miniature beta-sheet protein for targeting beta-amyloid oligomers implicated in Alzheimer's disease. Circular dichroism spectroscopy, thermal denaturation experiments, and immunoglobulin binding assays established that our beta-amyloid-targeted miniature protein, TJ10, presents a well-folded thermostable beta-sheet. TJ10 was found to prevent beta-amyloid fibrillization at stoichiometric concentrations and was also an effective inhibitor at substoichiometric concentrations. Thus our results provide a new and potent beta-sheet chemical template for effectively targeting beta-amyloid while also demonstrating a general strategy for targeting proteins implicated in other amyloidogenic diseases.

Amino Acid Sequence↗

PubMed2006

A minimalist approach toward protein recognition by epitope transfer from functionally evolved beta-sheet surfaces.

New approaches for identifying small molecules that specifically target protein surfaces as opposed to active site clefts are of much current interest. Toward this goal, we describe a three-step methodology: in step one, we target a protein of interest by directed evolution of a small beta-sheet scaffold; in step two, we identify residues on the scaffold that are implicated in binding; and in step three, we transfer the chemical information from the beta-sheet to a small molecule mimic. As a case study, we targeted the proteolytic enzyme thrombin, involved in blood coagulation, utilizing a library of beta-sheet epitopes displayed on phage that were previously selected for conservation of structure. We found that the thrombin-binding, beta-sheet displaying mini-proteins retained their structure and stability while inhibiting thrombin at low micromolar inhibition constants. A conserved dityrosine recognition motif separated by 9.2 A was found to be common among the mini-protein inhibitors and was further verified by alanine scanning. A molecule containing two tyrosine residues separated by a linker that matched the spacing on the beta-sheet scaffold inhibited thrombin, whereas a similar dityrosine molecule separated by a shorter 6 A linker could not. Moreover, kinetic analysis revealed that both the mini-protein as well as its minimalist mimic with only two functional residues exhibited noncompetitive inhibition of thrombin. Thus, this reductionist approach affords a simple methodology for transferring information from structured protein scaffolds to yield small molecule leads for targeting protein surfaces with novel mechanisms of action.

Amino Acid Sequence↗

PubMed2006

Highly selective cyclic peptide ligands for NeutrAvidin and avidin identified by phage display.

Screening combinatorial libraries of conformationally constrained peptides against macromolecular targets is utilized in identifying novel drug leads and in developing new reagents for chemical biology. In methods such as phage-display selections, biotinylated macromolecular targets are often immobilized on avidin- and streptavidin-functionalized supports. Thus, the characterization of peptides that bind avidin and streptavidin is necessary for accurate interpretation of screening and selection results. Toward this goal, we panned a phage-displayed cyclic peptide library against NeutrAvidin, a chemically deglycosylated version of avidin. The selection produced a highly homologous consensus motif (Asp-Arg/Leu-Ala-Ser/Thr-Pro-Tyr/Trp). Two of these cyclic peptides, CDRATPYC and CDRASPYC, bound both NeutrAvidin and avidin with low-micromolar dissociation constants, whereas their acyclic counterparts had negligible affinity (< 80-fold). Moreover, these cyclic peptides were very specific for their targets and did not bind the structurally and functionally similar protein, streptavidin. Thus, we have identified a new class of cyclic peptides, distinct from the much-studied streptavidin-binding His-Pro-Gln peptide motif. These results will not only allow for discriminating between desired and background cyclic peptide motifs in selections and screens but also provide a new protein/peptide model system and a useful reagent in chemical biology that can have utility in protein immobilization, purification, and chemical tagging.

Amino Acid Motifs↗

PubMed2005

Single-site mutations in a hyperthermophilic variant of the B1 domain of protein G result in self-assembled oligomers.

We have characterized two homologous, single-point core mutants of a 57-residue, hyperthermophilic variant of the B1 domain of protein G (HTB1). These single-point mutations in HTB1 replace a Phe residue in the hydrophobic core with either a Glu or Asp residue. Both of these homologous core-variant mutants undergo significant structural rearrangement from the native, monomeric fold and exist as stable soluble oligomeric species of 5 and 30 nm in diameter. Gel-filtration, dynamic light scattering, circular dichroism spectroscopy, fluorescence spectroscopy, along with Congo Red and Thioflavin T binding clearly demonstrated that these core-variants undergo significant structural rearrangement from the native, monomeric ubiquitin fold. The two oligomeric species did not equilibrate over extended periods of time and displayed distinct secondary structures. The larger of the two species was found to possess structural features that are reminiscent of an emerging class of protein assemblies prone to beta-sheet-mediated aggregation. These results are significant as there are very few examples of extensive conformational or oligomerization switching brought about by single-point mutations in a stable protein-fold.

Amino Acid Sequence↗