A REDOR ssNMR Investigation of the Role of an N‑Terminus Lysine in R5 Silica Recognition

Diatoms are unicellular algae that construct cell walls called frustules by the precipitation of silica, using special proteins that order the silica into a wide variety of nanostructures. The diatom species <i>Cylindrotheca fusiformis</i> contains proteins called silaffins within its frustules, which are believed to assemble into supramolecular matrices that serve as both accelerators and templates for silica deposition. Studying the properties of these biosilicification proteins has allowed the design of new protein and peptide systems that generate customizable silica nanostructures, with potential generalization to other mineral systems. It is essential to understand the mechanisms of aggregation of the protein and its coprecipitation with silica. We continue previous investigations into the peptide R5, derived from silaffin protein sil1p, shown to independently catalyze the precipitation of silica nanospheres in vitro. We used the solid-state NMR technique ¹³C­{²⁹Si} and ¹⁵N­{²⁹Si} REDOR to investigate the structure and interactions of R5 in complex with coprecipitated silica. These experiments are sensitive to the strength of magnetic dipole–dipole interactions between the ¹³C nuclei in R5 and the ²⁹Si nuclei in the silica and thus yield distance between parts of R5 and ²⁹Si in silica. Our data show strong interactions and short internuclear distances of 3.74 ± 0.20 Å between ¹³CO Lys3 and silica. On the other hand, the C_α and C_β nuclei show little or no interaction with ²⁹Si. This selective proximity between the K3 CO and the silica supports a previously proposed mechanism of rapid silicification of the antimicrobial peptide KSL (KKVVFKVKFK) through an imidate intermediate. This study reports for the first time a direct interaction between the N-terminus of R5 and silica, leading us to believe that the N-terminus of R5 is a key component in the molecular recognition process and a major factor in silica morphogenesis

A Study of Phenylalanine Side-Chain Dynamics in Surface-Adsorbed Peptides Using Solid-State Deuterium NMR and Rotamer Library Statistics

Extracellular matrix proteins adsorbed onto mineral surfaces exist in a unique environment where the structure and dynamics of the protein can be altered profoundly. To further elucidate how the mineral surface impacts molecular properties, we perform a comparative study of the dynamics of nonpolar side chains within the mineral-recognition domain of the biomineralization protein salivary statherin adsorbed onto its native hydroxyapatite (HAP) mineral surface versus the dynamics displayed by the native protein in the hydrated solid state. Specifically, the dynamics of phenylalanine side chains (viz., F7 and F14) located in the surface-adsorbed 15-amino acid HAP-recognition fragment (SN15: DpSpSEEKFLRRIGRFG) are studied using deuterium magic angle spinning (²H MAS) line shape and spin–lattice relaxation measurements. ²H NMR MAS spectra and <i>T</i>₁ relaxation times obtained from the deuterated phenylalanine side chains in free and HAP-adsorbed SN15 are fitted to models where the side chains are assumed to exchange between rotameric states and where the exchange rates and a priori rotameric state populations are varied iteratively. In condensed proteins, phenylalanine side-chain dynamics are dominated by 180° flips of the phenyl ring, i.e., the “π flip”. However, for both F7 and F14, the number of exchanging side-chain rotameric states increases in the HAP-bound complex relative to the unbound solid sample, indicating that increased dynamic freedom accompanies introduction of the protein into the biofilm state. The observed rotameric exchange dynamics in the HAP-bound complex are on the order of 5–6 × 10⁶ s^–1, as determined from the deuterium MAS line shapes. The dynamics in the HAP-bound complex are also shown to have some solution-like behavioral characteristics, with some interesting deviations from rotameric library statistics