Base Dynamics in the HhaI Protein Binding Site.

Protein-DNA interactions play an important role in numerous biological functions within the living cell. In many of these interactions, the DNA helix is significantly distorted upon protein-DNA complex formation. The HhaI restriction-modification system is one such system, where the methylation target is flipped out of the helix when bound to the methyltransferase. However, the base flipping mechanism is not well understood. The dynamics of the binding site of the HhaI methyltransferase and endonuclease (underlined) within the DNA oligomer [d(G1A2T3A4G5C6G7C8T9A10T11C12)]2 are studied using deuterium solid-state NMR (SSNMR). SSNMR spectra obtained from DNAs deuterated on the base of nucleotides within and flanking the [5'-GCGC-3']2 sequence indicate that all of these positions are structurally flexible. Previously, conformational flexibility within the phosphodiester backbone and furanose ring within the target sequence has been observed and hypothesized to play a role in the distortion mechanism. However, whether that distortion was occurring through an active or passive mechanism remained unclear. These NMR data demonstrate that although the [5'-GCGC-3']2 sequence is dynamic, the target cytosine is not passively flipping out of the double-helix on the millisecond-picosecond time scale. Additionally, although previous studies have shown that both the furanose ring and phosphodiester backbone experience a change in dynamics upon methylation, which may play a role in recognition and cleavage by the endonuclease, our observations here indicate that methylation has no effect on the dynamics of the base itself.

Binding of Dipeptides to Fatty Acid Membranes Explains Their Colocalization in Protocells but Does Not Select for Them Relative to Unjoined Amino Acids.

Dipeptides, which consist of two amino acids joined by a peptide bond, have been shown to have catalytic functions. This observation leads to fundamental questions relevant to the origin of life. How could peptides have become colocalized with the first protocells? Which structural features would have determined the association of amino acids and peptides with membranes? Could the association of dipeptides with protocell membranes have driven molecular evolution, favoring dipeptides over individual amino acids? Using pulsed-field gradient nuclear magnetic resonance, we find that several prebiotic amino acids and dipeptides bind to prebiotic membranes. For amino acids, the side chains and carboxylate contribute to the interaction. For dipeptides, the extent of binding is generally less than that of the constituent amino acids, implying that other mechanisms would be necessary to drive molecular evolution. Nevertheless, our results are consistent with a scheme in which the building blocks of the biological polymers colocalized with protocells prior to the emergence of RNA and proteins.

Trimethylation of the R5 Silica-Precipitating Peptide Increases Silica Particle Size by Redirecting Orthosilicate Binding.

The unmodified R5 peptide from silaffin in the diatom Cylindrotheca fusiformis rapidly precipitates silica particles from neutral aqueous solutions of orthosilicic acid. A range of post-translational modifications found in R5 contribute toward tailoring silica morphologies in a species-specific manner. We investigated the specific effect of R5 lysine side-chain trimethylation, which adds permanent positive charges, on silica particle formation. Our studies revealed that a doubly trimethylated R5K3,4me3 peptide has reduced maximum activity yet, surprisingly, generates larger silica particles. Molecular dynamics simulations of R5K3,4me3 binding by the precursor orthosilicate anion revealed that orthosilicate preferentially associates with unmodified lysine side-chain amines and the peptide N terminus. Thus, larger silica particles arise from reduced orthosilicate association with trimethylated lysine side chains and their redirection to the N terminus of the R5 peptide.

A Step toward Molecular Evolution of RNA: Ribose Binds to Prebiotic Fatty Acid Membranes, and Nucleosides Bind Better than Individual Bases Do.

A major challenge in understanding how biological cells arose on the early Earth is explaining how RNA and membranes originally colocalized. We propose that the building blocks of RNA (nucleobases and ribose) bound to self-assembled prebiotic membranes. We have previously demonstrated that the bases bind to membranes composed of a prebiotic fatty acid, but evidence for the binding of sugars has remained a technical challenge. Here, we used pulsed-field gradient NMR spectroscopy to demonstrate that ribose and other sugars bind to membranes of decanoic acid. Moreover, the binding of some bases is strongly enhanced when they are linked to ribose to form a nucleoside or - with the addition of phosphate - a nucleotide. This enhanced binding could have played a role in the molecular evolution leading to the production of RNA.

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.

Prebiotic amino acids bind to and stabilize prebiotic fatty acid membranes.

The membranes of the first protocells on the early Earth were likely self-assembled from fatty acids. A major challenge in understanding how protocells could have arisen and withstood changes in their environment is that fatty acid membranes are unstable in solutions containing high concentrations of salt (such as would have been prevalent in early oceans) or divalent cations (which would have been required for RNA catalysis). To test whether the inclusion of amino acids addresses this problem, we coupled direct techniques of cryoelectron microscopy and fluorescence microscopy with techniques of NMR spectroscopy, centrifuge filtration assays, and turbidity measurements. We find that a set of unmodified, prebiotic amino acids binds to prebiotic fatty acid membranes and that a subset stabilizes membranes in the presence of salt and Mg2+ Furthermore, we find that final concentrations of the amino acids need not be high to cause these effects; membrane stabilization persists after dilution as would have occurred during the rehydration of dried or partially dried pools. In addition to providing a means to stabilize protocell membranes, our results address the challenge of explaining how proteins could have become colocalized with membranes. Amino acids are the building blocks of proteins, and our results are consistent with a positive feedback loop in which amino acids bound to self-assembled fatty acid membranes, resulting in membrane stabilization and leading to more binding in turn. High local concentrations of molecular building blocks at the surface of fatty acid membranes may have aided the eventual formation of proteins.

Solid-State NMR and MD Study of the Structure of the Statherin Mutant SNa15 on Mineral Surfaces.

Elucidation of the structure and interactions of proteins at native mineral interfaces is key to understanding how biological systems regulate the formation of hard tissue structures. In addition, understanding how these same proteins interact with non-native mineral surfaces has important implications for the design of medical and dental implants, chromatographic supports, diagnostic tools, and a host of other applications. Here, we combine solid-state NMR spectroscopy, isotherm measurements, and molecular dynamics simulations to study how SNa15, a peptide derived from the hydroxyapatite (HAP) recognition domain of the biomineralization protein statherin, interacts with HAP, silica (SiO2), and titania (TiO2) mineral surfaces. Adsorption isotherms are used to characterize the binding affinity of SNa15 to HAP, SiO2, and TiO2. We also apply 1D 13C CP MAS, 1D 15N CP MAS, and 2D 13C-13C DARR experiments to SNa15 samples with uniformly 13C- and 15N-enriched residues to determine backbone and side-chain chemical shifts. Different computational tools, namely TALOS-N and molecular dynamics simulations, are used to deduce secondary structure from backbone and side-chain chemical shift data. Our results show that SNa15 adopts an α-helical conformation when adsorbed to HAP and TiO2, but the helix largely unravels upon adsorption to SiO2. Interactions with HAP are mediated in general by acidic and some basic amino acids, although the specific amino acids involved in direct surface interaction vary with surface. The integrated experimental and computational approach used in this study is able to provide high-resolution insights into adsorption of proteins on interfaces.

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.

Solid state deuterium NMR study of LKα14 peptide aggregation in biosilica.

In nature, organisms including diatoms, radiolaria, and marine sponges use proteins, long chain polyamines, and other organic molecules to regulate the assembly of complex silica-based structures. Here, the authors investigate structural features of small peptides, designed to mimic the silicifying activities of larger proteins found in natural systems. LKα14 (Ac-LKKLLKLLKKLLKL-C), an amphiphilic lysine/leucine repeat peptide with an α-helical secondary structure at polar/apolar interfaces, coprecipitates with silica to form nanospheres. Previous 13C magic angle spinning studies suggest that the tetrameric peptide bundles that LKα14 is known to form in solution may persist in the silica-complexed form, and may also function as catalysts and templates for silica formation. To further investigate LKα14 aggregation in silica, deuterium solid-state nuclear magnetic resonance (2H ssNMR) was used to establish how leucine side-chain dynamics differ in solid LKα14 peptides isolated from aqueous solution, from phosphate-buffered solution, and in the silica-precipitated states. Modeling the 2H ssNMR line shapes probed the mechanisms of peptide preaggregation and silica coprecipitation. The resulting NMR data indicates that the peptide bundles in silica preserve the hydrophobic interior that they display in the hydrated solid state. However, NMR data also indicate free motion of the leucine residues in silica, a condition that may result from structural deformation of the aggregates arising from interactions between the surface lysine side chains and the surrounding silica matrix.

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.

Ultraslow Domain Motions in HIV-1 TAR RNA Revealed by Solid-State Deuterium NMR.

Intrinsic motions may allow HIV-1 transactivation response (TAR) RNA to change its conformation to form a functional complex with the Tat protein, which is essential for viral replication. Understanding the dynamic properties of TAR necessitates determining motion on the intermediate nanosecond-to-microsecond time scale. To this end, we performed solid-state deuterium NMR line-shape and T1Z relaxation-time experiments to measure intermediate motions for two uridine residues, U40 and U42, within the lower helix of TAR. We infer global motions at rates of ∼105 s-1 in the lower helix, which are much slower than those in the upper helix (∼106 s-1), indicating that the two helical domains reorient independently of one another in the solid-state sample. These results contribute to the aim of fully describing the properties of functional motions in TAR RNA.

Diatom mimics: directing the formation of biosilica nanoparticles by controlled folding of lysine-leucine peptides.

Silaffins, long chain polyamines, and other biomolecules found in diatoms are involved in the assembly of a large number of silica nanostructures under mild, ambient conditions. Nanofabrication researchers have sought to mimic the diatom's biosilica production capabilities by engineering proteins to resemble aspects of naturally occurring biomolecules. Such mimics can produce monodisperse biosilica nanospheres, but in vitro production of the variety of intricate biosilica nanostructures that compose the diatom frustule is not yet possible. In this study we demonstrate how LK peptides, composed solely of lysine (K) and leucine (L) amino acids arranged with varying hydrophobic periodicities, initiate the formation of different biosilica nanostructures in vitro. When L and K residues are arranged with a periodicity of 3.5 the α-helical form of the LK peptide produces monodisperse biosilica nanospheres. However, when the LK periodicity is changed to 3.0, corresponding to a 310 helix, the morphology of the nanoparticles changes to elongated rod-like structures. ß-strand LK peptides with a periodicity of 2.0 induce wire-like silica morphologies. This study illustrates how the morphology of biosilica can be changed simply by varying the periodicity of polar and nonpolar amino acids.

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.

Elucidating molecular motion through structural and dynamic filters of energy-minimized conformer ensembles.

Complex RNA structures are constructed from helical segments connected by flexible loops that move spontaneously and in response to binding of small molecule ligands and proteins. Understanding the conformational variability of RNA requires the characterization of the coupled time evolution of interconnected flexible domains. To elucidate the collective molecular motions and explore the conformational landscape of the HIV-1 TAR RNA, we describe a new methodology that utilizes energy-minimized structures generated by the program "Fragment Assembly of RNA with Full-Atom Refinement (FARFAR)". We apply structural filters in the form of experimental residual dipolar couplings (RDCs) to select a subset of discrete energy-minimized conformers and carry out principal component analyses (PCA) to corroborate the choice of the filtered subset. We use this subset of structures to calculate solution T1 and T(1ρ) relaxation times for (13)C spins in multiple residues in different domains of the molecule using two simulation protocols that we previously published. We match the experimental T1 times to within 2% and the T(1ρ) times to within less than 10% for helical residues. These results introduce a protocol to construct viable dynamic trajectories for RNA molecules that accord well with experimental NMR data and support the notion that the motions of the helical portions of this small RNA can be described by a relatively small number of discrete conformations exchanging over time scales longer than 1 µs.

Preparation of RNA samples with narrow line widths for solid state NMR investigations.

Solid state NMR can provide detailed structural and dynamic information on biological systems that cannot be studied under solution conditions, and can investigate motions which occur with rates that cannot be fully studied by solution NMR. This approach has successfully been used to study proteins, but the application of multidimensional solid state NMR to RNA has been limited because reported line widths have been too broad to execute most multidimensional experiments successfully. A reliable method to generate spectra with narrow line widths is necessary to apply the full range of solid state NMR spectroscopic approaches to RNA. Using the HIV-1 transactivation response (TAR) RNA as a model, we present an approach based on precipitation with polyethylene glycol that improves the line width of (13)C signals in TAR from >6 ppm to about 1 ppm, making solid state 2D NMR studies of selectively enriched RNAs feasible at ambient temperature.

Direct observation of phenylalanine orientations in statherin bound to hydroxyapatite surfaces.

Extracellular biomineralization proteins such as salivary statherin control the growth of hydroxyapatite (HAP), the principal component of teeth and bones. Despite the important role that statherin plays in the regulation of hard tissue formation in humans, the surface recognition mechanisms involved are poorly understood. The protein-surface interaction likely involves very specific contacts between the surface atoms and the key protein side chains. This study demonstrates for the first time the power of combining near-edge X-ray absorption fine structure (NEXAFS) spectroscopy with element labeling to quantify the orientation of individual side chains. In this work, the 15 amino acid N-terminal binding domain of statherin has been adsorbed onto HAP surfaces, and the orientations of phenylalanine rings F7 and F14 have been determined using NEXAFS analysis and fluorine labels at individual phenylalanine sites. The NEXAFS-derived phenylalanine tilt angles have been verified with sum frequency generation spectroscopy.