Solution Nuclear Magnetic Resonance Techniques Research Articles

Structural Analysis of Oligosaccharides and Glycoconjugates Using NMR.

Carbohydrate chains play critical roles in cellular recognition and subsequent signal transduction in the nervous system. Furthermore, gangliosides are targets for various amyloidogenic proteins associated with neurodegenerative disorders. To better understand the molecular mechanisms underlying these biological phenomena, atomic views are essential to delineate dynamic biomolecular interactions. Nuclear magnetic resonance (NMR) spectroscopy provides powerful tools for studying structures, dynamics, and interactions of biomolecules at the atomic level. This chapter describes the basics of solution NMR techniques and their applications to the analysis of 3D structures and interactions of glycoconjugates in the nervous system.

Examining the Transient Dark State in Protein-Quantum Dot Interaction by Relaxation-Based Solution NMR.

We probed the "dark" state involved in the protein-quantum dot (QD) interaction using a relaxation-based solution nuclear magnetic resonance (NMR) approach. We examined the dynamics and exchange kinetics of the ubiquitin-CdTe model system, which undergoes a fast exchange in the transverse relaxation time scale. We applied the recently developed dark-state exchange saturation transfer (DEST), lifetime line broadening (ΔR2), and exchange-induced chemical shift (δex) solution NMR techniques to obtain a residue-specific binding behavior of the protein on the QD surface. The variation in the estimated 15N-R2bound values clearly shows the dynamic nature of bound Ub. Upon mapping the amino acid residues showing a faster relaxation rate on the electrostatic potential surface of the protein, we have determined that the interaction is preferably electrostatic, and the amino acid residues involved in binding lie on the positively charged surface of the protein. We believe that our experimental approach should provide more in-depth knowledge to engineer new hybrid protein-QD systems in the future.

NMR Spin Relaxation Theory of Biomolecules Undergoing Highly Asymmetric Exchange with Large Interaction Partners

The transient interactions of proteins and other molecules with much larger structures, such as synthetic or biological nanoparticles, lead to certain types of enhanced nuclear magnetic resonance (NMR) spin relaxation effects, which can be accurately measured by multidimensional solution NMR techniques. These relaxation effects provide new information about the nanostructures and the protein, their interactions, internal dynamics, and associated kinetic and thermodynamic parameters, such as exchange rates and populations. Although theoretical treatments exist that cover either the fast or slow exchange limits, a theoretical treatment that applies to all practically relevant exchange processes is still missing. A unified theoretical framework is presented for this purpose based on a stochastic Liouville equation (SLE). It covers nuclear spin dynamics, overall rotational diffusion of both the protein and the nanostructure, the exchange process between a free state and a bound state, and internal protein dynamics. Although the numerical implementation of the SLE typically involves large matrices, it is shown here that it is computationally still tractable for situations relevant in practice. Application of the theory demonstrates how transverse relaxation is substantially impacted by the kinetics of binding on a wide range of exchange timescales. It is further shown that when exchange occurs on the appropriate timescale, transverse relaxation is able to report on internal dynamics far slower than observable by traditional transverse relaxation experiments. The SLE will allow the realistic and quantitative interpretation of experimental NMR data reporting about transient protein-nanoparticle interactions, thereby providing a powerful tool for the characterization of protein dynamics modes on a vast range of timescales including motions that may be functionally relevant.

NMR-Guided Repositioning of Non-Steroidal Anti-Inflammatory Drugs into Tight Junction Modulators.

Bioavailability is a major bottleneck in the clinical application of medium molecular weight therapeutics, including protein and peptide drugs. Paracellular transport of these molecules is hampered by intercellular tight junction (TJ) complexes. Therefore, safe chemical regulators for TJ loosening are desired. Here, we showed a potential application of select non-steroidal anti-inflammatory drugs (NSAIDs) as TJ modulators. Based on our previous observation that diclofenac and flufenamic acid directly bound various PDZ domains with a broad specificity, we applied solution nuclear magnetic resonance techniques to examine the interaction of other NSAIDs and the first PDZ domain (PDZ1) of zonula occludens (ZO)-1, ZO-1(PDZ1). Inhibition of ZO-1(PDZ1) is expected to provide loosening of the epithelial barrier function because the domain plays a crucial role in maintaining TJ integrity. Accordingly, diclofenac and indomethacin were found to decrease the subcellular localization of claudin (CLD)-2 but not occludin and ZO-1 at the apicolateral intercellular compartment of Madin–Darby canine kidney (MDCK) II cells. These NSAIDs exhibited 125–155% improved paracellular efflux of fluorescein isothiocyanate insulin for the Caco-2 cell monolayer. We propose that these NSAIDs can be repurposed as drug absorption enhancers for peptide drugs.

The role of calcium in the interaction between calmodulin and a minimal functional construct of eukaryotic elongation factor 2 kinase.

Eukaryotic elongation factor 2 kinase (eEF-2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF-2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM-regulated kinases. We had previously identified a minimal construct of eEF-2K (TR) that is activated similarly to the wild-type enzyme by CaM in vitro and retains its ability to phosphorylate eEF-2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1-methyls of TR and Ile δ1- and Met ε-methyls of CaM, as probes of their mutual interaction and the influence of Ca2+ thereon. We find that in the absence of Ca2+ , CaM exclusively utilizes its C-terminal lobe (CaMC ) to engage the N-terminal CaM-binding domain (CBD) of TR in a high-affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca2+ -loaded N-terminal lobe of CaM (CaMN ) at increased Ca2+ levels serves to enhance the affinity further. These latter interactions under Ca2+ saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca2+ -free state. Our data are consistent with a scenario in which Ca2+ enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM-bound species without significantly modifying the conformation of TR within the final, active complex.

Unexpected Solubility Enhancement of Drug Bases in the Presence of a Dimethylaminoethyl Methacrylate Copolymer.

The methacrylate copolymer Eudragit EPO (EPO) has previously shown to greatly enhance solubilization of acidic drugs via ionic interactions and by multiple hydrophobic contacts with polymeric side chains. The latter type of interaction could also play a role for solubilization of other compounds than acids. The aim of this study was therefore to investigate the solubility of six poorly soluble bases in presence and absence of EPO by quantitative ultrapressure liquid chromatography with concomitant X-ray powder diffraction analysis of the solid state. For a better mechanistic understanding, spectra and diffusion data were obtained by 1H nuclear magnetic resonance (NMR) spectroscopy. Unexpected high solubility enhancement (up to 360-fold) was evidenced in the presence of EPO despite the fact that bases and polymer were both carrying positive charges. This exceptional and unexpected solubilization was not due to a change in the crystalline solid state. NMR spectra and measured diffusion coefficients indicated both strong drug-polymer interactions in the bulk solution, and diffusion data suggested conformational changes of the polymer in solution. Such conformational changes may have increased the accessibility and extent of hydrophobic contacts thereby leading to increased overall molecular interactions. These initially surprising solubilization results demonstrate that excipient selection should not be based solely on simple considerations of, for example, opposite charges of drug and excipient, but it requires a more refined molecular view. Different solution NMR techniques are especially promising tools to gain such mechanistic insights.

Structural analysis of oligosaccharides and glycoconjugates using NMR.

Carbohydrate chains play critical roles in cellular recognition and subsequent signal transduction in the nervous system. Furthermore, gangliosides are targets for various amyloidogenic proteins associated with neurodegenerative disorders. To better understand the molecular mechanisms underlying these biological phenomena, atomic views are essential to delineate dynamic biomolecular interactions. Nuclear magnetic resonance (NMR) spectroscopy provides powerful tools for studying structures, dynamics, and interactions of biomolecules at the atomic level. This chapter describes the basics of solution NMR techniques and their applications to the analysis of 3D structures and interactions of glycoconjugates in the nervous system.

Trapping the dynamic acyl carrier protein in fatty acid biosynthesis

Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis. Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain. ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway. The transient nature of ACP-enzyme interactions impose major obstacles to obtaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to study protein-protein interactions effectively. Here we describe the application of a mechanism-based probe that allows active site-selective covalent crosslinking of AcpP to FabA, the Escherichia coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase, respectively. We report the 1.9 Å crystal structure of the crosslinked AcpP-FabA complex as a homodimer in which AcpP exhibits two different conformations, representing probable snapshots of ACP in action: the 4'-phosphopantetheine group of AcpP first binds an arginine-rich groove of FabA, then an AcpP helical conformational change locks AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution nuclear magnetic resonance techniques, including chemical shift perturbations and residual dipolar coupling measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. These techniques, in combination with molecular dynamics simulations, show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies may be broadly applicable to fatty acid, polyketide and non-ribosomal biosynthesis. Here the foundation is laid for defining the dynamic action of carrier-protein activity in primary and secondary metabolism, providing insight into pathways that can have major roles in the treatment of cancer, obesity and infectious disease.

Low-ε Static Probe Development for15N-1H Solid-state NMR Study of Membrane Proteins for an 800 MHz NB Magnet

H heteronuclear dipolar coupling in dilute membrane proteins oriented in hydrated anddielectrically lossy lipid environments. The system employed a 800 MHz narrow-bore magnet. A solenoid coilstrip shield was used to reduce deleterious RF sample heating by minimizing the conservative electric fieldsgenerated by the double-tuned resonator at high magnetic fields. The probe's design, construction, andperformance in solid-state NMR experiments at high magnetic fields are described here. Such high-resolutionsolid-state NMR spectroscopic analysis of static oriented samples in hydrated phospholipid bilayers or bicellescould aid the structural analysis of dilute biological membrane proteins.Key Words : Solid-state NMR probe, Low- e, Probe design, Membrane proteins, BicellesIntroductionMembrane proteins are important in various essentialsystems and regulatory processes, ranging from the com-plicated network of cellular communications to the meta-bolism and breaking down of unwanted substances in thehuman body. Hundreds of diseases involve the mis-foldingor mis-assembling of integral membrane proteins and over60% of all drug molecules target membrane proteins.Despite their importance, these protein's intrinsic structure-function relationships are not understood as their three-dimensional structures have not been fully elucidated. Thestudy of membrane proteins by current methods is hamperedby the need of a native bilayer environment, which is diffi-cult using most biophysical techniques. They are notoriouslydifficult to crystallize for X-ray crystallography and theirslow reorientation rates when combined with lipids or deter-gents hinder the use of conventional solution NMR (NuclearMagnetic Resonance) techniques. Solid-state NMR spectroscopy is a relatively new meansof investigating the structures of solid biological samples.

Star mesogens — Synthesis and structural characterization using 1D and 2D solution NMR techniques and mesophase characterization

Novel star mesogens based on trimesic acid and symmetrical side arm cores with terminal alkoxy groups were synthesized via a divergent approach. The central core and side arms were connected through alkyl spacers. All the synthesized mesogens and their intermediates were characterized thoroughly using Fourier transform infrared (FT-IR), 1H nuclear magnetic resonance (NMR), 13C NMR, and mass spectrometers. One representative mesogen was subjected to the two-dimensional (2D) NMR experiments to ascertain the structure of the mesogens. The mesophase characterization was carried out using hot-stage optical polarizing microscopy (HOPM), differential scanning calorimetry (DSC), and X-ray diffraction (XRD) techniques. Many of the molecules with an ethyloxy spacer were found to be nonmesogenic, whereas all the molecules with a butyloxy spacer showed liquid crystalline phases. The increase of terminal chain length decreased the transition temperatures. The nematic phase was observed for the mesogens with short terminal chain length, whereas smectic polymorphism was observed on increasing the terminal chain length. The results of a variable temperature powder X-ray diffraction of the representative sample support the smectic layer ordering.

NMR studies of peptide T, an inhibitor of HIV infectivity, in an aqueous environment

The synthetic octapeptide peptide T (ASTTTNYT) has been shown to interfere with binding of the HIV-1 envelope glycoprotein gp120 to the chemokine receptor R5, thus preventing viral infection. This study investigated the degree of conformational order of two analogs of peptide T, one biologically active (D-Ala peptide T amide) and one inactive (D-Ala, D-Tyr peptide T amide) using nuclear magnetic resonance (NMR) spectroscopy in an aqueous environment, both in solution and in the frozen solid state. Standard solution NMR techniques such as DQFCOSY, HMQC, ROESY and inversion recovery measurements have been utilized to characterize these peptides. Solid state NMR experiments were likewise employed to study the peptides in a frozen glycerol:water mixture. The NMR results indicate that the monomeric form of both peptide T analogs have considerable conformational heterogeneity. Solid state NMR studies indicate aggregation of D-Ala peptide T, possibly into a beta-sheet structure, at concentrations higher than 10 mM.

Design, high-level expression, purification and characterization of soluble fragments of the hepatitis C virus NS3 RNA helicase suitable for NMR-based drug discovery methods and mechanistic studies.

RNA helicases represent a family of enzymes that unwind double-stranded (ds) RNA in a nucleoside triphosphate (NTP)-dependent fashion and which are required in all aspects of cellular RNA metabolism and processing. The hepatitis C virus (HCV) non-structural 3 (NS3) protein possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion of the 631 amino acid residue bifunctional enzyme. The HCV NS3 RNA helicase is of key importance in the life cycle of HCV, which makes it a target for the development of therapeutics. However, neither the precise mechanism nor the substrate structure has been defined for this enzyme. For nuclear magnetic resonance (NMR)-based drug discovery methods and for mechanistic studies we engineered, prepared and characterized various truncated constructs of the 451-residue HCV NS3 RNA helicase. Our goal was to produce smaller fragments of the enzyme, which would be amenable to solution NMR techniques while retaining their native NTP and/or nucleic acid binding sites. Solution conditions were optimized to obtain high-quality heteronuclear NMR spectra of nitrogen-15 isotope-labeled constructs, which are typical of well-folded monomeric proteins. Moreover, NMR binding studies and functional data directly support the correct folding of these fragments.

[15] Filamentous bacteriophage for aligning RNA, DNA, and proteins for measurement of nuclear magnetic resonance dipolar coupling interactions

Publisher Summary This chapter describes the application of filamentous bacteriophage as a versatile tool for generating partially ordered solutions of nucleic acids and proteins. The Pfl phage system described in the chapter is an ideal method for obtaining a tunable degree of alignment of RNA and DNA oligomers and some proteins in solution, which then allows the measurement of homonuclear and heteronuclear dipolar coupling interactions. The Pfl filamentous phage system is ideally suited for obtaining a tunable degree of alignment in RNA and DNA molecules and acidic proteins for the observation of dipolar couplings. The phage particles are fully aligned at magnetic fields as low as 300 MHz and are stable indefinitely at a range of temperatures and buffer conditions. These dipolar couplings provide long-range structural information that is presently unavailable by standard solution nuclear magnetic resonance (NMR) techniques; therefore, in the future, dipolar coupling data may prove to be as indispensable as 1 H– 1 H nuclear Overhauser effect (NOE) in the solution structure determinations of macromolecules. This should be especially true for structure determinations of RNA and DNA oligomers where the low density of protons compared to proteins leads to fewer useful NOE distance constraints, and therefore generally less well-defined structures. In addition, the current solution NMR techniques are generally unable to determine global structural features, such as bending in nucleic acids, due because of the local nature of the NOE distance constraints.

Porogens in the preparation of microporous hydrogels based on poly(ethylene oxides)

To enable the synthesis of hydrogels containing microporosity, a family of pore generating additives (porogens) were synthesized from poly(ethylene glycols) (PEGs) with different molecular weights using naphthyl acetic acid. The products formed were characterized by UV-vis, differential scanning calorimetry and solution nuclear magnetic resonance techniques. Subsequently, poly(ethylene oxide)-urethane hydrogels were synthesized incorporating the above mentioned PEG porogens to modify the structure of the hydrogel. These highly water soluble PEG porogens were inert and extracted out in water. The hydrogels obtained exhibited significant increase in the equilibrium water uptake. This was attributed to the formation of a microporous structure in the hydrogel. It was also evidenced by the observed increase in the diffusion coefficients of the drugs proxyphylline and vitamin B 12 through this hydrogel. The proportional increase was greater for the higher molecular weight vitamin B 12 than for the proxyphylline. These results may be useful in developing porous hydrogels for controlled release technology.