Biomedical subjects
Publications and source records attributed to Gary P Drobny.
Solution and solid-state NMR have been used conjointly to probe the internal motions of a DNA dodecamer containing the recognition site for the HhaI methyltransferase. The results strongly suggest that ns-mus motions contribute to the functionally relevant dynamic properties of nucleic acids during DNA methylation.
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 alpha-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.
Acidic proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), Ca10(PO4)6(OH)2, the main mineral component of bone and teeth. Key to understanding the structural basis of protein-crystal recognition and protein control of hard tissue growth is the nature of interactions between the protein side chains and the crystal surface. In an earlier work we have measured the proximity of the lysine (K6) side chain in an SN-15 peptide fragment of the salivary protein statherin adsorbed to the Phosphorus-rich surface of HAP using solid-state NMR recoupling experiments. 15N{31P} rotational echo double resonance (REDOR) NMR data on the side-chain nitrogen in K6 gave rise to three different models of protein-surface interaction to explain the experimental data acquired. In this work we extend the analysis of the REDOR data by examining the contribution of interactions between surface phosphorus atoms to the observed 15N REDOR decay. We performed 31P-31P recoupling experiments in HAP and (NH4)2HPO4 (DHP) to explore the nature of dipolar coupled 31P spin networks. These studies indicate that extensive networks of dipolar coupled 31P spins can be represented as stronger effective dipolar couplings, the existence of which must be included in the analysis of REDOR data. We carried out 15N{31P} REDOR in the case of DHP to determine how the size of the dephasing spin network influences the interpretation of the REDOR data. Although use of an extended 31P coupled spin network simulates the REDOR data well, a simplified 31P dephasing system composed of two spins with a larger dipolar coupling also simulates the REDOR data and only perturbs the heteronuclear couplings very slightly. The 31P-31P dipolar couplings between phosphorus nuclei in HAP can be replaced by an effective dipolar interaction of 600 Hz between two 31P spins. We incorporated this coupling and applied the above approach to reanalyze the 15N{31P} REDOR of the lysine side chain approaching the HAP surface and have refined the binding models proposed earlier. We obtain 15N-31P distances between 3.3 and 5 A from these models that are indicative of the possibility of a lysine-phosphate hydrogen bond.
Extracellular matrix proteins regulate hard tissue growth by acting as adhesion sites for cells, by triggering cell signaling pathways, and by directly regulating the primary and/or secondary crystallization of hydroxyapatite, the mineral component of bone and teeth. Despite the key role that these proteins play in the regulation of hard tissue growth in humans, the exact mechanism used by these proteins to recognize mineral surfaces is poorly understood. Interactions between mineral surfaces and proteins very likely involve specific contacts between the lattice and the protein side chains, so elucidation of the nature of interactions between protein side chains and their corresponding inorganic mineral surfaces will provide insight into the recognition and regulation of hard tissue growth. Isotropic chemical shifts, chemical shift anisotropies (CSAs), NMR line-width information, (13)C rotating frame relaxation measurements, as well as direct detection of correlations between (13)C spins on protein side chains and (31)P spins in the crystal surface with REDOR NMR show that, in the peptide fragment derived from the N-terminal 15 amino acids of salivary statherin (i.e., SN-15), the side chain of the phenylalanine nearest the C-terminus of the peptide (F14) is dynamically constrained and oriented near the surface, whereas the side chain of the phenylalanine located nearest to the peptide's N-terminus (F7) is more mobile and is oriented away from the hydroxyapatite surface. The relative dynamics and proximities of F7 and F14 to the surface together with prior data obtained for the side chain of SN-15's unique lysine (i.e., K6) were used to construct a new picture for the structure of the surface-bound peptide and its orientation to the crystal surface.
Magic angle spinning NMR techniques can be used to determine the molecular structure of proteins adsorbed onto polymer and mineral surfaces, but the degree to which the orientation of proteins on surfaces can be uniquely determined by NMR is less well understood. In this manuscript, REDOR data obtained from model systems are analyzed with a view to determine the orientation of rare spins coupled to a lattice populated by strongly coupled spin 1/2 nuclei. When the surface is populated by closely spaced spins, the REDOR dephasing of a rare spin on the protein contact point to the surface is under certain circumstances complicated by contributions from homonuclear dipolar interactions between the spins of the lattice. To study multiple spin effects on the dephasing signal in rotational-echo-double-resonance experiments, we carried out a measurement on crystalline diammonium hydrogen phosphate as a model for a spin system with multiple dipolar interactions. Information about the (31)P-(31)P interactions is gathered from the reference measurement in the experiment. To fit the experimental (15)N and (31)P dephasing data well, it was necessary to account for as many as 6 and 8 spins in simulations, respectively. Using a single spin-pair interaction with an unknown distance yielded a good fit to the (31)P data with a distance of 2.7A that is nearly an Angström shorter than the shortest distance in the crystal structure. Homonuclear couplings are shown to have a significant effect on the expected dephasing.
REDOR is a solid-state NMR technique frequently applied to biological structure problems. Through incorporation of phosphorothioate groups in the nucleic acid backbone and mono-fluorinated nucleotides, 31P{19F} REDOR has been used to study the binding of DNA to drugs and RNA to proteins through the detection of internuclear distances as large as 13-14 A. In this work, 31P{19F} REDOR is further refined for use in nucleic acids by the combined use of selective placement of phosphorothioate groups and the introduction of nucleotides containing trifluoromethyl (-CF3) groups. To ascertain the REDOR-detectable distance limit between an unique phosphorous spin and a trifluoromethyl group and to assess interference from intermolecular couplings, a series of model compounds and DNA dodecamers were synthesized each containing a unique phosphorous label and trifluoromethyl group or a single 19F nucleus. The dipolar coupling constants of the various 31P and 19F or -CF3 containing compounds were compared using experimental and theoretical dephasing curves involving several models for intermolecular interactions.
Hydroxyapatite (HAP) is the main mineral component of teeth. It is well-known that several salivary proteins and peptides bind strongly to HAP to regulate crystal growth. Interactions between a peptide derived from the N-terminal fragment of the salivary protein statherin and HAP were measured utilizing rotational-echo double-resonance (REDOR) nuclear magnetic resonance (NMR). The REDOR measurement from the side chain of the salivary peptide to the HAP surface is complicated by two effects: a possible additional dipolar coupling to a phosphorylated side chain and the potential proximity of phosphorus atoms to each other, resulting in a homonuclear dipolar interaction. Both of these effects were addressed, and the smallest model applicable to our system includes the nitrogen-15 (15N) spin in the lysine side chain and two phosphorus-31 (31P) spins, at least one of which must be from the surface phosphates of the HAP.
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.
We have performed solid-state 31P-19F REDOR nuclear magnetic resonance (NMR) experiments to monitor changes in minor groove width of the oligonucleotide d(CGCAAA2'FUTGGC)*d(GCCAAT(pS)TT GCG) (A3T2) upon binding of the drug distamycin A at different stoichiometries. In the hydrated solid-state sample, the minor groove width for the unbound DNA, measured as the 2'FU7-pS19 inter-label distance, was 9.4 +/- 0.7 A, comparable to that found for similar A:T-rich DNAs. Binding of a single drug molecule is observed to cause a 2.4 A decrease in groove width. Subsequent addition of a second drug molecule results in a larger conformational change, expanding this minor groove width to 13.6 A, consistent with the results of a previous solution NMR study of the 2:1 complex. These 31P-19F REDOR results demonstrate the ability of solid-state NMR to measure distances of 7-14 A in DNA-drug complexes and provide the first example of a direct spectroscopic measurement of minor groove width in nucleic acids.
Internal motions of d-ribose selectively 2H-labeled at the 2' position were measured using solid state 2H NMR experiments. A sample of d-ribose-2'-d was prepared in a hydrated, non-crystalline state to eliminate effects of crystal-packing. Between temperatures of -74 and -60 degrees C the C2'-H2' bond was observed to undergo two kinds of motions which were similar to those of C2'-H2'/H2" found previously in crystalline deoxythymidine (Hiyama et al. (1989) J. Am. Chem. Soc., 111, 8609-8613): (1) Nanosecond motion of small angular displacement with an apparent activation energy of 3.6+/-0.7 kcal mol(-1), and (2) millisecond to microsecond motion of large amplitude with an apparent activation energy > or =4 kcal mol(-1). At -74 degrees C, the slow, large-amplitude motion was best characterized as a two-site jump with a correlation time on the millisecond time scale, whereas at -60 degrees C it was diffusive on the microsecond time scale. The slow, large-amplitude motions of the C2'-H2' bond are most likely from interconversions between C2'-endo and C3'-endo by way of the O4'-endo conformation, whereas the fast, small-amplitude motions are probably librations of the C2'-H2' bond within the C2'-endo and C3'-endo potential energy minima.
Proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (bones/teeth) and calcium oxalate (kidney stones). Despite their importance in hard-tissue formation and remodeling, and in pathological processes such as stone formation and arterial calcification, there is little known of the protein structure-function relationships that govern hard-tissue engineering. Here we review early studies that have utilized solid-state NMR (ssNMR) techniques to provide in situ secondary-structure determination of statherin and statherin peptides on their biologically relevant hydroxyapatite (HAP) surfaces. In addition to direct structural study, molecular dynamics studies have provided considerable insight into the protein-binding footprint on hydroxyapatite. The molecular insight provided by these studies has also led to the design of biomimetic fusion peptides that utilize nature's crystal-recognition mechanism to display accessible and dynamic bioactive sequences from the HAP surface. These peptides selectively engage adhesion receptors and direct specific outside-in signaling pathway activation in osteoblast-like cells.
The adsorption or covalent attachment of biological macromolecules onto polymer materials to improve their biocompatibility has been pursued using a variety of approaches, but key to understanding their efficacy is the verification of the structure and dynamics of the immobilized biomolecules. Here we present data on peptides designed to adsorb from aqueous solutions onto highly porous hydrophobic surfaces with specific helical secondary structures. Small linear peptides composed of alternating leucine and lysine residues were synthesized, and their adsorption onto porous polystyrene surfaces was studied using a combination of solid-state NMR techniques. Using conventional solid-state NMR experiments and newly developed double-quantum techniques, their helical structure was verified. Large-amplitude dynamics on the NMR time scale were not observed, suggesting irreversible adsorption of the peptides. Their association, adsorption, and structure were examined as a function of helix length and sequence periodicity, and it was found that, at higher solution concentrations, peptides as short as seven amino acids adsorb with defined secondary structures. Two-dimensional double-quantum experiments using (13)C-enriched peptide sequences allow high-resolution determination of secondary structure in heterogeneous environments where the peptides are a minor component of the material. These results shed light on how polymeric surfaces may be surface-modified by structured peptides and demonstrate the level of molecular structural and dynamic information solid-state NMR can provide.