Combining Molecular and Spin Dynamics Simulations with Solid-State NMR: A Case Study of Amphiphilic Lysine-Leucine Repeat Peptide Aggregates.

Interpreting dynamics in solid-state molecular systems requires characterization of the potentially heterogeneous environmental contexts of molecules. In particular, the analysis of solid-state nuclear magnetic resonance (ssNMR) data to elucidate molecular dynamics (MD) involves modeling the restriction to overall tumbling by neighbors, as well as the concentrations of water and buffer. In this exploration of the factors that influence motion, we utilize atomistic MD trajectories of peptide aggregates with varying hydration to mimic an amorphous solid-state environment and predict ssNMR relaxation rates. We also account for spin diffusion in multiply spin-labeled (up to 19 nuclei) residues, with several models of dipolar-coupling networks. The framework serves as a general approach to determine essential spin couplings affecting relaxation, benchmark MD force fields, and reveal the hydration dependence of dynamics in a crowded environment. We demonstrate the methodology on a previously characterized amphiphilic 14-residue lysine-leucine repeat peptide, LKα14 (Ac-LKKLLKLLKKLLKL-c), which has an α-helical secondary structure and putatively forms leucine-burying tetramers in the solid state. We measure the R1 relaxation rates of uniformly 13C-labeled and site-specific 2H-labeled leucines in the hydrophobic core of LKα14 at multiple hydration levels. Studies of 9 and 18 tetramer bundles reveal the following: (a) for the incoherent component of 13C relaxation, the nearest-neighbor spin interactions dominate, while the 1H-1H interactions have minimal impact; (b) the AMBER ff14SB dihedral barriers for the leucine Cγ-Cδ bond ("methyl rotation barriers") must be lowered by a factor of 0.7 to better match the 2H data; (c) proton-driven spin diffusion explains some of the discrepancy between experimental and simulated rates for the Cß and Cα nuclei; and (d) 13C relaxation rates are mostly underestimated in the MD simulations at all hydrations, and the discrepancies identify likely motions missing in the 50 ns MD trajectories.

Serine-Lysine Peptides as Mediators for the Production of Titanium Dioxide: Investigating the Effects of Primary and Secondary Structures Using Solid-State NMR Spectroscopy and DFT Calculations.

A biomimetic approach to the formation of titania (TiO2) nanostructures is desirable because of the mild conditions required in this form of production. We have identified a series of serine-lysine peptides as candidates for the biomimetic production of TiO2 nanostructures. We have assayed these peptides for TiO2-precipitating activity upon exposure to titanium bis(ammonium lactato)dihydroxide and have characterized the resulting coprecipitates using scanning electron microscopy. A subset of these assayed peptides efficiently facilitates the production of TiO2 nanospheres. Here, we investigate the process of TiO2 nanosphere formation mediated by the S-K peptides KSSKK- and SKSK3SKS using one-dimensional and two-dimensional solid-state NMR (ssNMR) on peptide samples with uniformly 13C-enriched residues. ssNMR is used to assign 13C chemical shifts (CSs) site-specifically in each free peptide and TiO2-embedded peptide, which are used to derive secondary structures in the neat and TiO2 coprecipitated states. The backbone 13C CSs are used to assess secondary structural changes undergone during the coprecipitation process. Side-chain 13C CS changes are analyzed with density functional theory calculations and used to determine side-chain conformational changes that occur upon coprecipitation with TiO2 and to determine surface orientation of lysine side chains in TiO2-peptide composites.

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 Cylindrotheca fusiformis 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 Cα and Cß 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.

Comparative Study of Secondary Structure and Interactions of the R5 Peptide in Silicon Oxide and Titanium Oxide Coprecipitates Using Solid-State NMR Spectroscopy.

A biomimetic, peptide-mediated approach to inorganic nanostructure formation is of great interest as an alternative to industrial production methods. To investigate the role of peptide structure on silica (SiO2) and titania (TiO2) morphologies, we use the R5 peptide domain derived from the silaffin protein to produce uniform SiO2 and TiO2 nanostructures from the precursor silicic acid and titanium bis(ammonium lactato)dihydroxide, respectively. The resulting biosilica and biotitania nanostructures are characterized using scanning electron microscopy. To investigate the process of R5-mediated SiO2 and TiO2 formation, we carry out 1D and 2D solid-state NMR (ssNMR) studies on R5 samples with uniformly 13C- and 15N-labeled residues to determine the backbone and side-chain chemical shifts. 13C chemical shift data are in turn used to determine peptide backbone torsion angles and secondary structure for the R5 peptide neat, in silica, and in titania. We are thus able to assess the impact of the different mineral environments on peptide structure, and we can further elucidate from 13C chemical shifts change the degree to which various side chains are in close proximity to the mineral phases. These comparisons add to the understanding of the role of R5 and its structure in both SiO2 and TiO2 formation.

Orientation and conformation of osteocalcin adsorbed onto calcium phosphate and silica surfaces.

Adsorption isotherms, circular dichroism (CD) spectroscopy, x-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to investigate the adsorption of human osteocalcin (hOC) and decarboxylated (i.e., Gla converted back to Glu) hOC (dhOC) onto various calcium phosphate surfaces as well as silica surfaces. The adsorption isotherms and XPS nitrogen signals were used to track the amount of adsorbed hOC and dhOC. The intensities of key ToF-SIMS amino acid fragments were used to assess changes in the structure of adsorbed hOC and dhOC. CD spectra were used to investigate the secondary structure of OC. The largest differences were observed when the proteins were adsorbed onto silica versus calcium phosphate surfaces. Similar amounts (3-4 at. % N) of hOC and dhOC were adsorbed onto the silica surface. Higher amounts of hOC and dhOC were adsorbed on all the calcium phosphate surfaces. The ToF-SIMS data showed that the intensity of the Cys amino acid fragment, normalized to intensity of all amino acid fragments, was significantly higher (∼×10) when the proteins were adsorbed onto silica. Since in the native OC structure the cysteines are located in the center of three α-helices, this indicates both hOC and dhOC are more denatured on the silica surface. As hOC and dhOC denature upon adsorption to the silica surface, the cysteines become more exposed and are more readily detected by ToF-SIMS. No significant differences were detected between hOC and dhOC adsorbed onto the silica surface, but small differences were observed between hOC and dhOC adsorbed onto the calcium phosphate surfaces. In the OC structure, the α-3 helix is located above the α-1 and α-2 helices. Small differences in the ToF-SIMS intensities from amino acid fragments characteristic of each helical unit (Asn for α-1; His for α-2; and Phe for α-3) suggests either slight changes in the orientation or a slight uncovering of the α-1 and α-2 for adsorbed dhOC. XPS showed that similar amounts of hOC and dhOC were absorbed onto hydroxyapaptite and octacalcium phosphate surfaces, but ToF-SIMS detected some small differences in the amino acid fragment intensities on these surfaces for adsorbed hOC and dhOC.

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 ((2)H MAS) line shape and spin-lattice relaxation measurements. (2)H NMR MAS spectra and T1 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 × 10(6) 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.

Silica morphogenesis by lysine-leucine peptides with hydrophobic periodicity.

The use of biomimetic approaches in the production of inorganic nanostructures is of great interest to the scientific and industrial community due to the relatively moderate physical conditions needed. In this vein, taking cues from silaffin proteins used by unicellular diatoms, several studies have identified peptide candidates for the production of silica nanostructures. In the current article, we study intensively one such silica-precipitating peptide, LKα14 (Ac-LKKLLKLLKKLLKL-c), an amphiphilic lysine/leucine repeat peptide that self-organizes into an α-helical secondary structure under appropriate concentration and buffer conditions. The suggested mechanism of precipitation is that the sequestration of hydrophilic lysines on one side of this helix allows interaction with the negatively charged surface of silica nanoparticles, which in turn can aggregate further into larger structures. To investigate the process, we carry out 1D and 2D solid-state NMR (ssNMR) studies on samples with one or two uniformly (13)C- and (15)N-labeled residues to determine the backbone and side-chain chemical shifts. We also further study the dynamics of two leucine residues in the sequence through (13)C spin-lattice relaxation times (T1) to determine the impact of silica coprecipitation on their mobility. Our results confirm the α-helical secondary structure in both the neat and silica-complexed states of the peptide, and the patterns of chemical shift and relaxation time changes between the two states suggest possible mechanisms of self-aggregation and silica precipitation.

Deuterium magic angle spinning NMR used to study the dynamics of peptides adsorbed onto polystyrene and functionalized polystyrene surfaces.

LKα14 is a 14 amino acid peptide with a periodic sequence of leucine and lysine residues consistent with an amphipathic α-helix. This "hydrophobic periodicity" has been found to result in an α-helical secondary structure at air-water interfaces and on both polar and nonpolar solid polymer surfaces. In this paper, the dynamics of LKα14 peptides, selectively deuterated at a single leucine and adsorbed onto polystyrene and carboxylated polystyrene beads, are studied using (2)H magic angle spinning (MAS) solid state NMR over a 100 °C temperature range. We first demonstrate the sensitivity enhancement possible with (2)H MAS techniques, which in turn enables us to obtain high-quality (2)H NMR spectra for selectively deuterated peptides adsorbed onto solid polymer surfaces. The extensive literature shows that the dynamics of leucine side chains are sensitive to the local structural environment of the protein. Therefore, the degree to which the dynamics of leucine side chains and the backbone of the peptide LKα14 are influenced by surface proximity and surface chemistry is studied as a function of temperature with (2)H MAS NMR. It is found that the dynamics of the leucine side chains in LKα14 depend strongly upon the orientation of the polymer on the surface, which in turn depends on whether the LKα14 peptide adsorbs onto a polar or nonpolar surface. (2)H MAS line shapes therefore permit probes of surface orientation over a wide temperature range.

Sum frequency generation and solid-state NMR study of the structure, orientation, and dynamics of polystyrene-adsorbed peptides.

The power of combining sum frequency generation (SFG) vibrational spectroscopy and solid-state nuclear magnetic resonance (ssNMR) spectroscopy to quantify, with site specificity and atomic resolution, the orientation and dynamics of side chains in synthetic model peptides adsorbed onto polystyrene (PS) surfaces is demonstrated in this study. Although isotopic labeling has long been used in ssNMR studies to site-specifically probe the structure and dynamics of biomolecules, the potential of SFG to probe side chain orientation in isotopically labeled surface-adsorbed peptides and proteins remains largely unexplored. The 14 amino acid leucine-lysine peptide studied in this work is known to form an alpha-helical secondary structure at liquid-solid interfaces. Selective, individual deuteration of the isopropyl group in each leucine residue was used to probe the orientation and dynamics of each individual leucine side chain of LKalpha14 adsorbed onto PS. The selective isotopic labeling methods allowed SFG analysis to determine the orientations of individual side chains in adsorbed peptides. Side chain dynamics were obtained by fitting the deuterium ssNMR line shape to specific motional models. Through the combined use of SFG and ssNMR, the dynamic trends observed for individual side chains by ssNMR have been correlated with side chain orientation relative to the PS surface as determined by SFG. This combination provides a more complete and quantitative picture of the structure, orientation, and dynamics of these surface-adsorbed peptides than could be obtained if either technique were used separately.

A solid-state deuterium NMR and sum-frequency generation study of the side-chain dynamics of peptides adsorbed onto surfaces.

The artificial amphiphilic peptide LKalpha14 adopts a helical structure at interfaces, with opposite orientation of its leucine (L, hydrophobic) and lysine (K, hydrophilic) side chains. When peptides are adsorbed onto surfaces, different residue side chains necessarily have different proximities to the surface, depending on both their position in the helix and the composition of the surface itself. Deuterating the individual leucine residues (isopropyl-d(7)) permits the use of solid-state deuterium NMR spectroscopy as a site-specific probe of side-chain dynamics. In conjunction with sum-frequency generation as a probe of the peptide-binding face, we demonstrate that the mobility of specific leucine side chains at the interface is quantifiable in terms of their surface proximity.

Monitoring tat peptide binding to TAR RNA by solid-state 31P-19F REDOR NMR.

Complexes of the HIV transactivation response element (TAR) RNA with the viral regulatory protein tat are of special interest due in particular to the plasticity of the RNA at this binding site and to the potential for therapeutic targeting of the interaction. We performed REDOR solid-state NMR experiments on lyophilized samples of a 29 nt HIV-1 TAR construct to measure conformational changes in the tat-binding site concomitant with binding of a short peptide comprising the residues of the tat basic binding domain. Peptide binding was observed to produce a nearly 4 A decrease in the separation between phosphorothioate and 2'F labels incorporated at A27 in the upper helix and U23 in the bulge, respectively, consistent with distance changes observed in previous solution NMR studies, and with models showing significant rearrangement in position of bulge residue U23 in the bound-form RNA. In addition to providing long-range constraints on free TAR and the TAR-tat complex, these results suggest that in RNAs known to undergo large deformations upon ligand binding, 31P-19F REDOR measurements can also serve as an assay for complex formation in solid-state samples. To our knowledge, these experiments provide the first example of a solid-state NMR distance measurement in an RNA-peptide complex.