Donghua H. Zhou

High-performance solvent suppression for proton detected solid-state NMR

High-sensitivity proton detected experiments in solid-state NMR have been recently demonstrated in proton diluted proteins as well as fully protonated samples under fast magic-angle spinning. One key element for performing successful proton detection is effective solvent suppression achieved by pulsed field gradients (PFG) and/or saturation pulses. Here we report a high-performance solvent suppression method that attenuates multiple solvent signals simultaneously by more than a factor of 10,000, achieved by an optimized combination of homospoil gradients and supercycled saturation pulses. This method, which we call Multiple Intense Solvent Suppression Intended for Sensitive Spectroscopic Investigation of Protonated Proteins, Instantly (MISSISSIPPI), can be applied without a PFG probe. It opens up new opportunities for two-dimensional heteronuclear correlation spectroscopy of hydrated proteins at natural abundance as well as high-sensitivity and multi-dimensional experimental investigation of protein-solvent interactions.

Conformation-specific Binding of α-Synuclein to Novel Protein Partners Detected by Phage Display and NMR Spectroscopy

α-Synuclein (AS) is an intrinsically unstructured protein in aqueous solution but is capable of forming β-sheet-rich fibrils that accumulate as intracytoplasmic inclusions in Parkinson disease and certain other neurological disorders. However, AS binding to phospholipid membranes leads to a distinct change in protein conformation, stabilizing an extended amphipathic α-helical domain reminiscent of the exchangeable apolipoproteins. To better understand the significance of this conformational change, we devised a novel bacteriophage display screen to identify protein binding partners of helical AS and have identified 20 proteins with roles in diverse cellular processes related to membrane trafficking, ion channel modulation, redox metabolism, and gene regulation. To verify that the screen identifies proteins with specificity for helical AS, we further characterized one of these candidates, endosulfine α (ENSA), a small cAMP-regulated phosphoprotein implicated in the regulation of insulin secretion but also expressed abundantly in the brain. We used solution NMR to probe the interaction between ENSA and AS on the surface of SDS micelles. Chemical shift perturbation mapping experiments indicate that ENSA interacts specifically with residues in the N-terminal helical domain of AS in the presence of SDS but not in aqueous buffer lacking SDS. The ENSA-related protein ARPP-19 (cAMP-regulated phosphoprotein 19) also displays specific interactions with helical AS. These results confirm that the helical N terminus of AS can mediate specific interactions with other proteins and suggest that membrane binding may regulate the physiological activity of AS in vivo.

Rapid Analysis of Organic Compounds by Proton‐Detected Heteronuclear Correlation NMR Spectroscopy with 40 kHz Magic‐Angle Spinning

High-resolution magic-angle spinning (MAS) solid-state NMR (SSNMR) spectroscopy is a powerful tool for analyzing the structural and dynamical properties of organic compounds; for example, unique insights into solid phase polymorphism/pseudo-polymorphism, hydrogen bonding, and stereoisomerism have been achieved,[1, 2] and these results apply to important classes of pharmaceutical solids in both pure and dosage forms.[3] Most studies have employed simple one-dimensional 13C spectra, assigning resonances in comparison to solution state spectra.[3, 4] Unambiguous assignments are usually not possible when the solid and solution spectra differ substantially. Spectral editing experiments, which distinguish carbon types (C, CH, CH2, CH3), aid in confirming some assignments but are not always reliable, for example when significant molecular dynamics are present.[3]

Proton‐Detected Solid‐State NMR Spectroscopy of Natural‐Abundance Peptide and Protein Pharmaceuticals

The natural way: A sensitive NMR spectroscopic method is developed to obtain well-resolved two-dimensional spectra ((15)N-(1)H and (13)C-(1)H) for natural-abundance (that is, without the need for isotopic enrichment) large-molecule samples, such as biopharmaceuticals. This method gives structural insights on two lyophilized aprotinin samples and three insulin samples in lyophilized, microcrystalline suspension formulation (red; see picture) and fibril (green) forms.

Solution Structure and DNA-binding Properties of the Winged Helix Domain of the Meiotic Recombination HOP2 Protein.

Background: HOP2 protein promotes recombination and is required for meiotic chromosome synapsis. Results: The N terminus of HOP2 has a winged head DNA binding structure. Conclusion: The solution structure of the winged head DNA binding domain integrates biochemical and functional aspects of HOP2 recombinational function. Significance: Determining the three-dimensional structure of HOP2 is crucial to understand the mechanism of HOP2 action. The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination.

Membrane attachment and structure models of lipid storage droplet protein 1.

Neutral lipid triglycerides, a main reserve for fat and energy, are stored in organelles called lipid droplets. The storage and release of triglycerides are actively regulated by several proteins specific to the droplet surface, one of which in insects is PLIN1. PLIN1 plays a key role in the activation of triglyceride hydrolysis upon phosphorylation. However, the structure of PLIN1 and its relation to functions remain elusive due to its insolubility and crystallization difficulty. Here we report the first solid-state NMR study on the Drosophila melanogaster PLIN1 in combination with molecular dynamics simulation to show the structural basis for its lipid droplet attachment. NMR spin diffusion experiments were consistent with the predicted membrane attachment motif of PLIN1. The data indicated that PLIN1 has close contact with the terminal methyl groups of the phospholipid acyl chains. Structure models for the membrane attachment motif were generated based on hydrophobicity analysis and NMR membrane insertion depth information. Simulated NMR spectra from a trans-model agreed with experimental spectra. In this model, lipids from the bottom leaflet were very close to the surface in the region enclosed by membrane attachment motif. This may imply that in real lipid droplet, triglyceride molecules might be brought close to the surface by the same mechanism, ready to leave the droplet in the event of lipolysis. Juxtaposition of triglyceride lipase structure to the trans-model suggested a possible interaction of a conserved segment with the lipase by electrostatic interactions, opening the lipase lid to expose the catalytic center.

High-resolution proton CRAMPS NMR using narrowband analog filters and postponed data acquisition

Proton linewidths decrease with increasing magic-angle spinning (MAS) rates. However, without spin dilution by deuteration, even with the fastest MAS rates available today, the narrowest proton linewidths are obtained by using the combined rotation and multiple pulse spectroscopy (CRAMPS) method. Direct observation under windowed CRAMPS typically introduces several tens of times more noise, partly because wideband analog filters (e.g. 5 MHz) must be used or sometimes even bypassed. Here we report that it is possible to keep using narrowband analog filters (about 50 kHz cutoff frequency) in CRAMPS by taking advantage of the time delay caused by the filters, which is inversely proportional to the cutoff frequency. This delay coincides with typical CRAMPS cycle times, enabling acquisition of the data point in the next detection window. The noise of such CRAMPS spectra is only about 5 times larger than MAS-only spectra. This new method allows CRAMPS to be performed on systems that lack wideline hardware (wideband filters and fast ADCs), for example, older spectrometers originally intended for solution NMR.

Analyses of mineral specific surface area and hydroxyl substitution for intact bone.

Bone minerals possess two primary hydrogen sources: hydroxide ions in the nanocrystalline core and structural water in the amorphous surface layer. In order to accurately measure their concentrations using hydrogen to phosphorus cross polarization NMR spectroscopy, it is necessary to analyze the dependence of signal intensities on serial contact times, namely, cross polarization kinetics. A reliable protocol is developed to iteratively decompose the severely overlapped spectra and to analyze the cross-polarization kinetics, leading to measurement of hydroxyl and structural water concentrations. Structural water concentration is used to estimate mineral specific surface area and nanocrystal thickness for intact bone.

Wing 1 of protein HOP2 is as important as helix 3 in DNA binding by MD simulation

The repair of programmed DNA double-strand breaks through recombination is required for proper association and disjunction of the meiotic homologous chromosomes. Meiosis-specific protein HOP2 plays essential roles in recombination by promoting recombinase activities. The N-terminal domain of HOP2 interacts with DNA through helix 3 (H3) and wing 1 (W1). Mutations in wing 1 (Y65A/K67A/Q68A) slightly weakened the binding but mutations in helices 2 and 3 (Q30A/K44A/K49A) nearly abolished the binding. To better understand such differential effects at atomic level, molecular dynamics simulations were employed. Despite losing some hydrogen bonds, the W1-mutant DNA complex was rescued by stronger hydrophobic interactions. For the wild type and W1-mutant, the protein was found to slide along the DNA grooves as the DNA rolls along its double-helix axis. This motion could be functionally important to facilitate the precise positioning of the single-stranded DNA with the homologous double-stranded DNA. The sliding motion was reduced in the W1-mutant. The H-mutant nearly lost all intermolecular interactions. Moreover, an additional mutation in wing 1 (Y65A/K67A/Q68A/K69A) also caused complete complex dissociation. Therefore, both wing 1 and helix 3 make important contribution to the DNA binding, which could be important to the strand invasion function of HOP2 homodimer and HOP2-MND1 heterodimer. Similar to cocking a medieval crossbow with the archer’s foot placed in the stirrup, wing 1 may push the minor groove to cause distortion while helix 3 grabs the major groove.

Activity of oxygen-versus sulfur-containing analogs of the Flex-Het anticancer agent SHetA2

Five series of chromans with urea and thiourea linkers connecting a chroman unit (ring A) and a 4-substituted benzene unit (ring B) have been prepared and evaluated relative to SHetA2 (NSC 721689) for activity against the human A2780 ovarian cancer cell line. The lead compound SHetA2 had a sulfur in place of the oxygen in ring A and a thiourea linker to ring B. The 2-Me-4-Me series (two sets of geminal dimethyl groups at C2 and at C4 on the ring A unit) permitted direct comparison with SHetA2. Ring B in this series was evaluated with specific functional groups at C4 on the ring, including NO2, CO2Et, CF3, OCF3, CN and SO2NH2. The 2-H-4-Me series (only one geminal dimethyl group at the C4 position on ring A) permitted structure-activity relationship analysis to assess the importance of the hydrophobic geminal dimethyl groups on ring A to the activity of SHetA2. The remaining three series 2-Et-4-Me, 2-Me-4-Et and 2-Et-4-Et (ring A methyl groups replaced with ethyls at C2, at C4 and at both C2 and C4, respectively) offered the opportunity to modulate the hydrophobicity of the chroman moiety. Additionally, in all these series, the influence of a urea versus a thiourea linker was also investigated. The results of these modifications are summarized below. The exact analog of SHetA2 with oxygen substituted for sulfur in ring A (2a) showed comparable efficacy but a significantly lower IC50 against the ovarian cancer cell line. The urea linked analogs bearing CN, CF3 and OCF3 at C4 of ring B (3c,d and f) showed greater efficacy than SHetA2, but also had lower IC50 values. Removing the geminal dimethyl group at C2 (4a-c, 5a-c) caused a significant lowering of the efficacy and percent growth inhibition, indicating that the hydrophobic geminal dimethyl group at C2 in ring A is crucial for activity. Finally, replacing the geminal dimethyl groups with geminal diethyls on ring A in the urea derivatives gave 6b-c, 7c-d and 8b, all of which outperformed SHetA2 with respect to efficacy and IC50. The results for compounds 4-8 are in concurrence with modeling studies, which predicted that greater hydrophobicity in ring A would be beneficial. Binding energies were determined for compounds docked in silico to mortalin, the protein identified as a receptor of SHetA2. The urea linker promoted activity comparable to or, in some cases, greater than compounds with a thiourea linker. Several compounds achieved 94% efficacy and an IC50 of 2 μM, which were better than SHetA2 (84%, 3 μM).