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.

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 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 × 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.

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 T1ρ relaxation times for 13C 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 T1ρ 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.

Nature has evolved sophisticated strategies for engineering hardtissues through the interaction of proteins, and ultimately cells, with inorganic mineral phases. This process, called biomineralization, is how living organisms transform inorganic materials such as hydroxyapatite, calcite, and silica into highly intricate and organized structures. The remarkable material properties of shell, bone, and teeth come from the activities of proteins that function at the organic–inorganic interface. A better understanding of the biomolecular mechanisms used to promote or retard the formation of mineral-based structures could provide important design principles for the development of calcification inhibitors and promoters in orthopedics, cardiology, urology, and dentistry. With the knowledge of the structural basis for control of hard tissue growth by proteins, scientists could potentially develop materials using biomimetic principles with applications in catalysis, biosensors, electronic devices, and chromatographic separations, to name a few. Additionally, biomineralization also has potential applications in electronics, catalysis, magnetism, sensory devices, and mechanical design. Where man-made hard materials require the use of extreme temperatures, high pressure, and pH, biological organisms can accomplish these feats at ambient temperature and at physiological pH.Despite the fact that many researchers want to identify and control the structure of proteins at material and biomineral interfaces, there is a decided lack of molecular-level structure information available for proteins at biomaterial interfaces in general. In particular, this holds for mammalian proteins that directly control calcification processes in hard tissue. The most fundamental questions regarding the secondary and tertiary structures of proteins adsorbed to material surfaces, how proteins catalyze the formation of biomineral composites, or how proteins interact at biomaterial interfaces remain unanswered. This is largely due to a lack of methods capable of providing high-resolution structural information for proteins adsorbed to material surfaces under physiologically relevant conditions.In this Account, we highlight recent work that is providing insight into the structure and crystal recognition mechanisms of a salivary protein model system, as well as the structure and interactions of a peptide that catalyzes the formation of biosilica composites. To develop a better understanding of the structure and interactions of proteins in biomaterials, we have used solid-state NMR techniques to determine the molecular structure and dynamics of proteins and peptides adsorbed onto inorganic crystal surfaces and embedded within biomineral composites. This work adds to the understanding of the structure and crystal recognition mechanisms of an acidic human salivary phosphoprotein, statherin.

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 2H magic angle spinning (MAS) solid state NMR over a 100 °C temperature range. We first demonstrate the sensitivity enhancement possible with 2H MAS techniques, which in turn enables us to obtain high-quality 2H 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 2H 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. 2H MAS line shapes therefore permit probes of surface orientation over a wide temperature range.

Solution NMR spectroscopy can elucidate many features of the structure and dynamics of macromolecules, yet relaxation measurements, the most common source of experimental information on dynamics, can sample only certain ranges of dynamic rates. A complete characterization of motion of a macromolecule thus requires the introduction of complementary experimental approaches. Solid-state NMR spectroscopy successfully probes the time scale of nanoseconds to microseconds, a dynamic window where solution NMR results have been deficient, and probes conditions where the averaging effects of rotational diffusion of the molecule are absent. Combining the results of the two distinct techniques within a single framework provides greater insight into dynamics, but this task requires the common interpretation of results recorded under very different experimental conditions. Herein, we provide a unified description of dynamics that is robust to the presence of large-scale conformational exchange, where the diffusion tensor of the molecule varies on a time scale comparable to rotational diffusion in solution. We apply this methodology to the HIV-1 TAR RNA molecule, where conformational rearrangements are both substantial and functionally important. The formalism described herein is of greater generality than earlier combined solid-state/solution NMR interpretations, if detailed molecular structures are available, and can offer a more complete description of RNA dynamics than either solution or solid-state NMR spectroscopy alone.

The complex of the HIV TAR RNA with the viral regulatory protein Tat is of considerable interest, but the plasticity of this interaction has made it impossible so far to establish the structure of that complex. In order to explore a new approach to obtain structural information on protein−RNA complexes, we performed 13C/15N−19F REDOR NMR experiments in the solid state on TAR bound to a peptide comprising the RNA-binding section of Tat. A critical arginine in the peptide was uniformly 13C and 15N labeled, and 5-fluorouridine was incorporated at the U23 position of TAR. REDOR irradiation resulted in dephasing of the 13C and 15N resonances, indicating the proximity of the U23(5F)−C and U23(5F)−N spin pairs. Best fits to the REDOR data show the U23(5F)−C distances and the U23(5F)−N distances are in good agreement with the distances obtained from solution NMR structures of partial complexes of Tat with TAR. These results demonstrate that it is possible to study protein−RNA complexes using solid-state REDOR NMR measurements, adding to a growing list of solid state techniques for studying protein−nucleic acid complexes.

Functional RNA molecules are conformationally dynamic and sample a multitude of dynamic modes over a wide range of frequencies. Thus, a comprehensive description of RNA dynamics requires the inclusion of a broad range of motions across multiple dynamic rates which must be derived from multiple spectroscopies. Here we describe a slow conformational exchange theoretical approach to combining the description of local motions in RNA that occur in the nanosecond to microsecond window and are detected by solid-state NMR with nonrigid rotational motion of the HIV-1 transactivation response element (TAR) RNA in solution as observed by solution NMR. This theoretical model unifies the experimental results generated by solution and solid-state NMR and provides a comprehensive view of the dynamics of HIV-1 TAR RNA, a well-known paradigm of an RNA where function requires extensive conformational rearrangements. This methodology provides a quantitative atomic level view of the amplitudes and rates of the local and collective displacements of the TAR RNA molecule and provides directly motional parameters for the conformational capture hypothesis of this classical RNA−ligand interaction.

The artificial amphiphilic peptide LKα14 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-d7) 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.

The side chain carboxyl groups of acidic proteins found in the extra-cellular matrix (ECM) of mineralized tissues play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), the principal mineral component of bone and teeth. Among the acidic proteins found in the saliva is statherin, a 43-residue tyrosine-rich peptide that is a potent lubricant in the salivary pellicle and an inhibitor of both HAP crystal nucleation and growth. Three acidic amino acids—D1, E4, and E5—are located in the N-terminal 15 amino acid segment, with a fourth amino acid, E26, located outside the N-terminus. We have utilized 13C{31P} REDOR NMR to analyze the role played by acidic amino acids in the binding mechanism of statherin to the HAP surface by measuring the distance between the δ-carboxyl 13C spins of the three glutamic acid side chains of statherin (residues E4, E5, E26) and 31P spins of the phosphate groups at the HAP surface. 13C{31P} REDOR studies of glutamic-5-13C acid incorporated at positions E4 and E26 indicate a 13C−31P distance of more than 6.5 Å between the side chain carboxyl 13C spin of E4 and the closest 31P in the HAP surface. In contrast, the carboxyl 13C spin at E5 has a much shorter 13C−31P internuclear distance of 4.25 ± 0.09 Å, indicating that the carboxyl group of this side chain interacts directly with the surface. 13C T1ρ and slow-spinning MAS studies indicate that the motions of the side chains of E4 and E5 are more restricted than that of E26. Together, these results provide further insight into the molecular interactions of statherin with HAP surfaces.

Both solid-state and solution NMR relaxation measurements are routinely used to quantify the internal dynamics of biomolecules, but in very few cases have these two techniques been applied to the same system, and even fewer attempts have been made so far to describe the results obtained through these two methods through a common theoretical framework. We have previously collected both solution 13C and solid-state 2H relaxation measurements for multiple nuclei within the furanose rings of several nucleotides of the DNA sequence recognized by HhaI methyltransferase. The data demonstrated that the furanose rings within the GCGC recognition sequence are very flexible, with the furanose rings of the cytidine, which is the methylation target, experiencing the most extensive motions. To interpret these experimental results quantitatively, we have developed a dynamic model of furanose rings based on the analysis of solid-state 2H line shapes. The motions are modeled by treating bond reorientations as Brownian excursions within a restoring potential. By applying this model, we are able to reproduce the rates of 2H spin−lattice relaxation in the solid and 13C spin−lattice relaxation in solution using comparable restoring force constants and internal diffusion coefficients. As expected, the 13C relaxation rates in solution are less sensitive to motions that are slower than overall molecular tumbling than to the details of global molecular reorientation, but are somewhat more sensitive to motions in the immediate region of the Larmor frequency. Thus, we conclude that the local internal motions of this DNA oligomer in solution and in the hydrated solid state are virtually the same, and we validate an approach to the conjoint analysis of solution and solid-state NMR relaxation and line shapes data, with wide applicability to many biophysical problems.

Statherin is an enamel pellicle protein that inhibits hydroxyapatite (HAP) nucleation and growth, lubricates the enamel surface, and is recognized by oral bacteria in periodontal diseases. We report here from solid-state NMR measurements that the protein's C-terminal region folds into an α-helix upon adsorption to HAP crystals. This region contains the binding sites for bacterial fimbriae that mediate bacterial cell adhesion to the surface of the tooth. The helical segment is shown through long-range distance measurements to fold back onto the intermediate region (residues Y16–P28) defining the global fold of the protein. Statherin, previously shown to be unstructured in solution, undergoes conformation selection on its substrate mineral surface. This surface-induced folding of statherin can be related to its functionality in inhibiting HAP crystal growth and can explain how oral pathogens selectively recognize HAP-bound statherin.

Protein-biomineral interactions are paramount to materials production in biology, including the mineral phase of hard tissue. Unfortunately, the structure of biomineral-associated proteins cannot be determined by X-ray crystallography or solution nuclear magnetic resonance (NMR). Here we report a method for determining the structure of biomineral-associated proteins. The method combines solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. In addition, the algorithm is able to identify lattice geometries most compatible with ssNMR constraints, representing a quantitative, novel method for investigating crystal-face binding specificity. We use this method to determine most of the structure of human salivary statherin interacting with the mineral phase of tooth enamel. Computation and experiment converge on an ensemble of related structures and identify preferential binding at three crystal surfaces. The work represents a significant advance toward determining structure of biomineral-adsorbed protein using experimentally biased structure prediction. This method is generally applicable to proteins that can be chemically synthesized.Graphical AbstractDownload high-res image (285KB)Download full-size imageHighlights► Novel method for determining the structure of protein-absorbed states ► Method combines solid-state NMR and computational structure prediction ► Partial high-resolution structure of human salivary statherin binding tooth enamel ► Statherin binds hydroxyapatite via a highly charged helical-binding domain

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. β-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.