John J. Ferrie

Thioamide Substitution Selectively Modulates Proteolysis and Receptor Activity of Therapeutic Peptide Hormones

Peptide hormones are attractive as injectable therapeutics and imaging agents, but they often require extensive modification by mutagenesis and/or chemical synthesis to prevent rapid in vivo degradation. Alternatively, the single-atom, O-to-S modification of peptide backbone thioamidation has the potential to selectively perturb interactions with proteases while preserving interactions with other proteins, such as target receptors. Here, we use the validated diabetes therapeutic, glucagon-like peptide-1 (GLP-1), and the target of clinical investigation, gastric inhibitory polypeptide (GIP), as proof-of-principle peptides to demonstrate the value of thioamide substitution. In GLP-1 and GIP, a single thioamide near the scissile bond renders these peptides up to 750-fold more stable than the corresponding oxopeptides toward cleavage by dipeptidyl peptidase 4, the principal regulator of their in vivo stability. These stabilized analogues are nearly equipotent with their parent peptide in cyclic AMP activation assays, but the GLP-1 thiopeptides have much lower β-arrestin potency, making them novel agonists with altered signaling bias. Initial tests show that a thioamide GLP-1 analogue is biologically active in rats, with an in vivo potency for glycemic control surpassing that of native GLP-1. Taken together, these experiments demonstrate the potential for thioamides to modulate specific protein interactions to increase proteolytic stability or tune activation of different signaling pathways.

Potential Artifacts in Sample Preparation Methods Used for Imaging Amyloid Oligomers and Protofibrils due to Surface-Mediated Fibril Formation

Accurate imaging of nanometer-sized structures and morphologies is essential to characterizing amyloid species formed at various stages of amyloid aggregation. In this article, we examine the effect of different drying procedures on the final morphology of surface-mediated fibrils formed during the incubation period, which may then be mistaken as oligomers or protofibrils intentionally formed in solution for a particular study. Atomic force microscopy results show that some artifacts, such as globules, flakelike structures, and even micrometer-long fibrils, can be produced under various drying conditions. We also demonstrate that one can prevent drying artifacts by using an appropriate spin-coating procedure to dry amyloid samples. This procedure can bypass the wetting/dewetting transition of the liquid layer during the drying process and preserve the structure of interest on the substrate without generating drying artifacts.

Alpha Synuclein Fibrils Contain Multiple Binding Sites for Small Molecules

The fibrillary aggregation of the protein alpha synuclein (Asyn) is a hallmark of Parkinsons disease, and the identification of small molecule binding sites on fibrils is essential to the development of diagnostic imaging probes. A series of molecular modeling, photoaffinity labeling, mass spectrometry, and radioligand binding studies were conducted on Asyn fibrils. The results of these studies revealed the presence of three different binding sites within fibrillar Asyn capable of binding small molecules with moderate to high affinity. A knowledge of the amino acid residues in these binding sites will be important in the design of high affinity probes capable of imaging fibrillary species of Asyn.

Systematic Evaluation of Soluble Protein Expression Using a Fluorescent Unnatural Amino Acid Reveals No Reliable Predictors of Tolerability

Improvements in genetic code expansion have made preparing proteins with diverse functional groups almost routine. Nonetheless, unnatural amino acids (Uaas) pose theoretical burdens on protein solubility, and determinants of position-specific tolerability to Uaas remain underexplored. To broadly examine associations, we systematically assessed the effect of substituting the fluorescent Uaa, acridonylalanine, at more than 50 chemically, evolutionarily, and structurally diverse residues in two bacterial proteins: LexA and RecA. Surprisingly, properties that ostensibly contribute to Uaa tolerability-such as conservation, hydrophobicity, or accessibility-demonstrated no consistent correlations with resulting protein solubility. Instead, solubility is closely dependent on the location of the substitution within the overall tertiary structure, suggesting that intrinsic properties of protein domains, and not individual positions, are stronger determinants of Uaa tolerability. Consequently, those who seek to install Uaas in new target proteins should consider broadening, rather than narrowing, the types of residues screened for Uaa incorporation.

Investigating the Trimethylamine N-Oxide (TMAO) Induced Structure of α-Synuclein

Trimethylamine N-oxide (TMAO) is a naturally occurring osmolyte that is known to stabilize protein structure. Previous studies have shown that the addition of TMAO can induce folding of thermodynamically unstable proteins, causing them to regain high functional activity. In solution, monomeric α-synuclein (αS) is intrinsically disordered. Our laboratory and others have shown that αS undergoes significant compaction in the presence of TMAO. Previously, we have demonstrated that p-cyanophenylalanine and a thioamide can serve as a minimally perturbing probe pair for Forster Resonance Energy Transfer (FRET) experiments. Despite the utility of this pair in measuring short intramolecular distances, inclusion of the thioamide is synthetically intensive, rendering it difficult to generate a large library of double-labeled mutants for FRET studies. As an alternative, we have expressed a library of double-labeled αS mutants containing the genetically encodable FRET pair, Cnf and tryptophan (Trp). This set of double-labeled proteins will allow us to obtain a more comprehensive description of the TMAO-induced morphology of αS.