A peptide that inhibits hydroxyapatite growth is in an extended conformation on the crystal surface

Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solid-state NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constant-composition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two 13C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 ± 0.1 Å. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete α-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an α-helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.

Native display of complete foreign protein domains on the surface of hepatitis B virus capsids

The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence. Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes. The immunodominant c/e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence. Because of its central position, however, any c/e1 insert disrupts the core protein’s primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation. According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces. We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently. Using the green fluorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms fluorescent particles; hence, all of its structurally important parts must be properly folded. We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP. Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles. Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy.