Gary P. Drobny

Optimization of two-dimensional homonuclear relayed coherence transfer NMR spectroscopy

Abstract The optimization of the recently introduced two-dimensional homonuclear RELAY experiment is described. Practical guidelines for optimization of experimental parameters and data processing are presented. The effectiveness of relayed magnetization transfer is found to be strongly dependent on the type of spin system. Explicit calculations predict the relative intensities of RELAY cross peaks for a number of commonly occurring systems. The feasibility of doubly relayed magnetizatioin transfer experiments is discussed and demonstrated for a sample of gramacidin-S and for the trinucleotides A2′--5′A2′-5A.

Solution Structure of a Cisplatin-Induced DNA Interstrand Cross-Link

The widely used antitumor drug cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) reacts with DNA, cross-linking two purine residues through the N7 atoms, which reside in the major groove in B-form DNA. The solution structure of the short duplex [d(CATAGCTATG)]2 cross-linked at the GC:GC site was determined by nuclear magnetic resonance (NMR). The deoxyguanosine-bridging cis-diammineplatinum(II) lies in the minor groove, and the complementary deoxycytidines are extrahelical. The double helix is locally reversed to a left-handed form, and the helix is unwound and bent toward the minor groove. These findings were independently confirmed by results from a phase-sensitive gel electrophoresis bending assay. The NMR structure differs markedly from previously proposed models but accounts for the chemical reactivity, the unwinding, and the bending of cis-DDP interstrand cross-linked DNA and may be important in the formation and repair of these cross-links in chromatin.

Fourier transform multiple quantum nuclear magnetic resonance

The excitation and detection of multiple quantum transitions in systems of coupled spins offers, among other advantages, an increase in resolution over single quantum n.m.r. since the number of lines decreases as the order of the transition increases. This paper reviews the motivation for detecting multiple quantum transitions by a Fourier transform experiment and describes an experimental approach to high resolution multiple quantum spectra in dipolar systems along with results on some protonated liquid crystal systems. A simple operator formalism for the essential features of the time development is presented and some applications in progress are discussed.

Windowless dipolar recoupling: the detection of weak dipolar couplings between spin 12 nuclei with large chemical shift anisotropies

A new homonuclear dipolar recoupling technique is described which uses a sequence of phase-shifted, windowless irradiations applied synchronously with sample spinning. Experiments performed on a series of doubly labeled dicarboxylic acids, alanine-1,3-13C2, and 2′-deoxythymidine-4,6-13C2 demonstrate that this new windowless dipolar recoupling pulse sequence can accurately determine internuclear distances from polycrystalline solids in cases where the coupled spins have large chemical shift anisotropies and large differences in isotropic chemical shift.

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 α-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 LKα14 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.

Chimeric Peptides of Statherin and Osteopontin That Bind Hydroxyapatite and Mediate Cell Adhesion

Extracellular matrix proteins play key roles in controlling the activities of osteoblasts and osteoclasts in bone remodeling. These bone-specific extracellular matrix proteins contain amino acid sequences that mediate cell adhesion, and many of the bone-specific matrix proteins also contain acidic domains that interact with the mineral surface and may orient the signaling domains. Here we report a fusion peptide design that is based on this natural approach for the display of signaling peptide sequences at biomineral surfaces. Salivary statherin contains a 15-amino acid hydroxyapatite binding domain (N15) that is loosely helical in solution. To test whether N15 can serve to orient active peptide sequences on hydroxyapatite, the RGD and flanking residues from osteopontin were fused to the C terminus. The fusion peptides bound tightly to hydroxyapatite, and the N15-PGRGDS peptide mediated the dose-dependent adhesion of Moαv melanoma cells when immobilized on the hydroxyapatite surface. Experiments with an integrin-sorted Moαv subpopulation demonstrated that the αvβ3 integrin was the primary receptor target for the fusion peptide. Solid state NMR experiments showed that the RGD portion of the hydrated fusion peptide is highly dynamic on the hydroxyapatite surface. This fusion peptide framework may thus provide a straightforward design for immobilizing bioactive sequences on hydroxyapatite for biomaterials, tissue engineering, and vaccine applications.

Molecular recognition at the protein-hydroxyapatite interface.

Proteins found in mineralized tissues act as natures 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 natures 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.

Determination of local structure in solid nucleic acids using double quantum nuclear magnetic resonance spectroscopy

A theoretical analysis of dipolar recoupling with a windowless multipulse irradiation (DRAWS) is presented. Analytical expressions that describe the degree to which the DRAWS pulse sequence recouples the dipolar interaction as a function of offset and spinning rate are derived using Floquet theory. Numerical methods are used to assess the performance of DRAWS in the preparation and detection of multiple quantum coherence. Simulations indicate that the mutual orientation of two or more CSA tensors can be obtained with high accuracy from double quantum spectra prepared and detected by DRAWS irradiation (DQDRAWS). These expectations are born out by experiment and in particular, the mutual orientation of three 13C CSA tensors in selectively labeled 2-deoxythymidine are determined from DQDRAWS data. The results of the DQDRAWS analysis of CSA tensor orientation in 2-deoxythymidine are shown to be in excellent agreement with results obtained by conventional methods. Using these CSA tensor orientations and an ind...

Folding of the C-terminal bacterial binding domain in statherin upon adsorption onto hydroxyapatite crystals

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 proteins 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.

The theory of oscillator-coupled magnetic resonance with potential applications to molecular imaging

This article describes systems in which the precession of a single particle spin is magnetically coupled to the excitation of an oscillator. The behavior of such systems resembles that of a ‘‘folded’’ Stern–Gerlach experiment, in which the linear spatial trajectory of the original Stern–Gerlach experiment is folded into the cyclic trajectory of an oscillator. Both quantum and semiclassical solutions to the equations of motion are derived. The results encompass any kind of oscillator which couples to a magnetic field. Examples include mechanical cantilevers with a magnetic source affixed to them, as well as inductor‐capacitor resonant circuits. One potential application of oscillator‐coupled magnetic resonance is the imaging of biological molecules. Some design issues relevant to molecular imaging are discussed.