HPMA copolymer-d-aspartic acid octapeptide (D-Asp8) conjugates have been found to target the entire skeleton after systemic administration. In a recent study using the ovariectomized rat model of osteoporosis, we surprisingly discovered that D-Asp8 would favorably recognize resorption sites in skeletal tissues, while another bone-targeting moiety, alendronate (ALN), directs the delivery system to both formation and resorption sites. Atomic force microscopy (AFM) analyses reveal that ALN has a stronger binding force to hydroxyapatite (HA) than D-Asp8. In vitro HA binding studies indicate that D-Asp8 is more sensitive to change of HA crystallinity than ALN. Because the bone apatite in the newly formed bone (formation sites) usually has lower crystallinity than the resorption sites (mainly mature bone), we believe that the favorable recognition of D-Asp8 to the bone resorption sites could be attributed to its relatively weak binding to apatite, when compared to bisphosphonates, and the different levels of crystallinity of bone apatite at different functional domains of the skeleton.

Self-assembly of misfolded proteins into ordered fibrillar structures is a fundamental property of a wide range of proteins and peptides. This property is also linked with the development of various neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Environmental conditions modulate the misfolding and aggregation processes. We used a peptide, CGNNQQNY, from yeast prion protein Sup35, as a model system to address effects of environmental conditions on aggregate formation. GNNQQNY peptide self-assembles in fibrils with structural features that are similar to amyloidogenic proteins. Atomic Force Microscopy (AFM) and Thioflavin T (ThT) fluorescence assay were employed to follow the aggregation process at various pHs and ionic strengths. We also used single molecule AFM force spectroscopy to probe interactions between the peptides under various conditions. The ThT fluorescence data showed that the peptide aggregates fast at pH values approaching the peptide isoelectric point (pI=5.3) and the kinetics is 10 times slower at acidic pH (pH 2.0) suggesting that electrostatic interactions contribute to the peptide self-assembly into aggregates. This hypothesis was tested by the experiments performed at low (11 mM) and high (150 mM) ionic strengths. Indeed, the aggregation lag time measured at pH 2 at low ionic strength (11 mM) is 195 hours, whereas the lag time decreases ~5 times when ionic strength is increased to 150 mM. At conditions close to the pI value, pH 5.6, the aggregation lag time is 12 ± 6 hours under low ionic strength, and there is minimal change to the lag time at 150 mM NaCl. Ionic strength also influences the morphology of aggregates visualized with AFM. In pH 2.0 and at high ionic strength, the aggregates are twofold taller than those formed at low ionic strength. In parallel, AFM force spectroscopy studies revealed minimal contribution of electrostatics on dissociation of transient peptide dimers.

Assembly of amyloid proteins into aggregates requires the ordering of the monomers in oligomers and especially in such highly organized structures as fibrils. This ordering is accompanied by structural transitions leading to the formation of ordered β-structural motifs in proteins and peptides lacking secondary structures. To characterize the effect of the monomers arrangements on the aggregation process at various stages, we performed comparative studies of the yeast prion protein Sup35 heptapeptide (GNNQQNY) along with its dimeric form CGNNQQNY-(d-Pro)-G-GNNQQNY. The (d-Pro)-G linker in this construct is capable of adopting a β-turn, facilitating the assembly of the dimer into the dimeric antiparallel hairpin structure (AP-hairpin). We applied Atomic Force Microscopy (AFM) techniques to follow peptide-peptide interactions at the single molecule level, to visualize the morphology of aggregates formed by both constructs, thioflavin T (ThT) fluorescence to follow the aggregation kinetics, and circular dichroism (CD) spectroscopy to characterize the secondary structure of the constructs. The ThT fluorescence data showed that the AP-hairpin aggregation kinetics is insensitive to the external environment such as ionic strength and pH contrary to the monomers the kinetics of which depends dramatically on the ionic strength and pH. The AFM topographic imaging revealed that AP--hairpins primarily assemble into globular aggregates, whereas linear fibrils are primary assemblies of the monomers suggesting that both constructs follow different aggregation pathways during the self-assembly. These morphological differences are in line with the AFM force spectroscopy experiments and CD spectroscopy measurements, suggesting that the AP-hairpin is structurally rigid regardless of changes of environmental factors.