T. Gopinath

Direct observation of the three regions in α-synuclein that determine its membrane-bound behaviour

α-synuclein (αS) is a protein involved in neurotransmitter release in presynaptic terminals, and whose aberrant aggregation is associated with Parkinson’s disease. In dopaminergic neurons, αS exists in a tightly regulated equilibrium between water-soluble and membrane-associated forms. Here we used a combination of solid-state and solution-state NMR spectroscopy to characterize the conformations of αS bound to lipid membranes mimicking the composition and physical properties of synaptic vesicles. The study evidences three αS regions possessing distinct structural and dynamical properties, including an N-terminal helical segment having a role of membrane-anchor, an unstructured C-terminal region that is weakly associated with the membrane, and a central region acting as a sensor of the lipid properties and determining the affinity of αS membrane binding. Taken together, our data define the nature of the interactions of αS with biological membranes and provide insights into their roles in the function and in the molecular processes leading the aggregation of this protein.

Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban

Significance The sarcoplasmic reticulum Ca2+-ATPase (SERCA)/phospholamban complex regulates cardiac muscle contractility by controlling Ca2+ transport from the cytosol to the lumen of the sarcoplasmic reticulum. By mapping the interactions between these two membrane proteins, we found that SERCA function depends on the equilibria between transient conformational states of phospholamban. Phosphorylation of phospholamban shifts the equilibria, enhancing SERCA function. This mechanism explains why tuning phospholamban’s structural dynamics can modulate SERCA function and may aid in designing innovative therapeutic approaches to heart failure. The membrane protein complex between the sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLN) controls Ca2+ transport in cardiomyocytes, thereby modulating cardiac contractility. β-Adrenergic-stimulated phosphorylation of PLN at Ser-16 enhances SERCA activity via an unknown mechanism. Using solid-state nuclear magnetic resonance spectroscopy, we mapped the physical interactions between SERCA and both unphosphorylated and phosphorylated PLN in membrane bilayers. We found that the allosteric regulation of SERCA depends on the conformational equilibrium of PLN, whose cytoplasmic regulatory domain interconverts between three different states: a ground T state (helical and membrane associated), an excited R state (unfolded and membrane detached), and a B state (extended and enzyme-bound), which is noninhibitory. Phosphorylation at Ser-16 of PLN shifts the populations toward the B state, increasing SERCA activity. We conclude that PLN’s conformational equilibrium is central to maintain SERCA’s apparent Ca2+ affinity within a physiological window. This model represents a paradigm shift in our understanding of SERCA regulation by posttranslational phosphorylation and suggests strategies for designing innovative therapeutic approaches to enhance cardiac muscle contractility.

Dual Acquisition Magic‐Angle Spinning Solid‐State NMR‐Spectroscopy: Simultaneous Acquisition of Multidimensional Spectra of Biomacromolecules

Fast data collection: a general method for dual data acquisition of multidimensional magic-angle spinning solid-state NMR experiments is presented. The method uses a simultaneous Hartmann-Hahn cross-polarization from (1)H to (13)C and (15)N nuclei and exploits the long-living (15)N polarization for parallel acquisition of two multidimensional experiments.

Sensitivity enhancement in static solid-state NMR experiments via single- and multiple-quantum dipolar coherences.

We present a new method for enhancing the sensitivity in static solid-state NMR experiments for a gain in signal-to-noise ratio of up to 40%. This sensitivity enhancement is different from the corresponding solution NMR sensitivity enhancement schemes and is achieved by combining single- and multiple-quantum dipolar coherences. While this new approach is demonstrated for the polarization inversion spin exchange at magic angle (PISEMA) experiment, it can be generalized to the other separated local field experiments for solid-state NMR spectroscopy. This method will have a direct impact on solid-state NMR spectroscopy of liquid crystals as well as of membrane proteins aligned in lipid membranes.

Role of the Strength of Drug-Polymer Interactions on the Molecular Mobility and Crystallization Inhibition in Ketoconazole Solid Dispersions

The effects of specific drug-polymer interactions (ionic or hydrogen-bonding) on the molecular mobility of model amorphous solid dispersions (ASDs) were investigated. ASDs of ketoconazole (KTZ), a weakly basic drug, with each of poly(acrylic acid) (PAA), poly(2-hydroxyethyl methacrylate) (PHEMA), and polyvinylpyrrolidone (PVP) were prepared. Drug-polymer interactions in the ASDs were evaluated by infrared and solid-state NMR, the molecular mobility quantified by dielectric spectroscopy, and crystallization onset monitored by differential scanning calorimetry (DSC) and variable temperature X-ray diffractometry (VTXRD). KTZ likely exhibited ionic interactions with PAA, hydrogen-bonding with PHEMA, and weaker dipole-dipole interactions with PVP. On the basis of dielectric spectroscopy, the α-relaxation times of the ASDs followed the order: PAA > PHEMA > PVP. In addition, the presence of ionic interactions also translated to a dramatic and disproportionate decrease in mobility as a function of polymer concentration. On the basis of both DSC and VTXRD, an increase in strength of interaction translated to higher crystallization onset temperature and a decrease in extent of crystallization. Stronger drug-polymer interactions, by reducing the molecular mobility, can potentially delay the crystallization onset temperature as well as crystallization extent.

3D DUMAS: simultaneous acquisition of three-dimensional magic angle spinning solid-state NMR experiments of proteins.

Using the DUMAS (Dual acquisition Magic Angle Spinning) solid-state NMR approach, we created new pulse schemes that enable the simultaneous acquisition of three dimensional (3D) experiments on uniformly (13)C, (15)N labeled proteins. These new experiments exploit the simultaneous cross-polarization (SIM-CP) from (1)H to (13)C and (15)N to acquire two 3D experiments simultaneously. This is made possible by bidirectional polarization transfer between (13)C and (15)N and the long living (15)N z-polarization in solid state NMR. To demonstrate the power of this approach, four 3D pulse sequences (NCACX, CANCO, NCOCX, CON(CA)CX) are combined into two pulse sequences (3D DUMAS-NCACX-CANCO, 3D DUMAS-NCOCX-CON(CA)CX) that allow simultaneous acquisition of these experiments, reducing the experimental time by approximately half. Importantly, the 3D DUMAS-NCACX-CANCO experiment alone makes it possible to obtain the majority of the backbone sequential resonance assignments for microcrystalline U-(13)C,(15)N ubiquitin. The DUMAS approach is general and applicable to many 3D experiments, nearly doubling the performance of NMR spectrometers.

Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers.

Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring 1H-15N dipolar couplings (DC) and 15N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles’ heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([1H,15N]-SE-PISEMA-PDSD). The incorporation of 2D 15N/15N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the 15N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.

Sensitivity Enhancement of Separated Local Field Experiments: Application to Membrane Proteins

Separated local field (SLF) experiments have been used for almost three decades to obtain structural information in solid-state NMR. These experiments resolve chemical shift anisotropy (CSA) from dipole-dipole interactions (dipolar couplings, DC) in isolated spin systems. Both CSA and DC data can be converted into orientational constraints to elucidate the secondary structure and topology of membrane proteins in oriented lipid bilayers. Here, we propose a new suite of sensitivity enhanced SLF pulse sequences to measure CSA and DC for aligned membrane proteins and liquid crystalline molecules that will decrease the time needed for data acquisition. We demonstrate the efficacy of these new sensitivity enhanced experiments using both a single crystal of N-acetyl leucine and a single pass membrane protein sarcolipin reconstituted in aligned lipid bicelles. These results lay the groundwork for the routine application of this methodology for studying the structure and topology of membrane proteins.

On the performance of spin diffusion NMR techniques in oriented solids: prospects for resonance assignments and distance measurements from separated local field experiments.

NMR spin diffusion experiments have the potential to provide both resonance assignment and internuclear distances for protein structure determination in oriented solid-state NMR. In this paper, we compared the efficiencies of three spin diffusion experiments: proton-driven spin diffusion (PDSD), cross-relaxation-driven spin diffusion (CRDSD), and proton-mediated proton transfer (PMPT). As model systems for oriented proteins, we used single crystals of N-acetyl-L-(15)N-leucine (NAL) and N-acetyl-L-(15)N-valyl-L-(15)N-leucine (NAVL) to probe long and short distances, respectively. We demonstrate that, for short (15)N/(15)N distances such as those found in NAVL (3.3 Å), the PDSD mechanism gives the most intense cross-peaks, while, for longer distances (>6.5 Å), the CRDSD and PMPT experiments are more efficient. The PDSD was highly inefficient for transferring magnetization across distances greater than 6.5 Å (NAL crystal sample), due to small (15)N/(15)N dipolar couplings (<4.5 Hz). Interestingly, the mismatched Hartmann-Hahn condition present in the PMPT experiment gave more intense cross-peaks for lower (1)H and (15)N RF spinlock amplitudes (32 and 17 kHz, respectively) rather than higher values (55 and 50 kHz), suggesting a more complex magnetization transfer mechanism. Numerical simulations are in good agreement with the experimental findings, suggesting a combined PMPT and CRDSD effect. We conclude that, in order to assign SLF spectra and measure short- and long-range distances, the combined use of homonuclear correlation spectra, such as the ones surveyed in this work, are necessary.

Sensitivity and resolution enhancement of oriented solid-state NMR: Application to membrane proteins

Oriented solid-state NMR (O-ssNMR) spectroscopy is a major technique for the high-resolution analysis of the structure and topology of transmembrane proteins in native-like environments. Unlike magic angle spinning (MAS) techniques, O-ssNMR spectroscopy requires membrane protein preparations that are uniformly oriented (mechanically or magnetically) so that anisotropic NMR parameters, such as dipolar and chemical shift interactions, can be measured to determine structure and orientation of membrane proteins in lipid bilayers. Traditional sample preparations involving mechanically aligned lipids often result in short relaxation times which broaden the (15)N resonances and encumber the manipulation of nuclear spin coherences. The introduction of lipid bicelles as membrane mimicking systems has changed this scenario, and the more favorable relaxation properties of membrane protein (15)N and (13)C resonances make it possible to develop new, more elaborate pulse sequences for higher spectral resolution and sensitivity. Here, we describe our recent progress in the optimization of O-ssNMR pulse sequences. We explain the theory behind these experiments, demonstrate their application to small and medium size proteins, and describe the technical details for setting up these new experiments on the new generation of NMR spectrometers.