Structure and dynamics of membrane peptides from solid-state NMR

Solid-state NMR is among the most important analytical techniques to provide atomic-level structural and dynamic information of chemical and biological systems. Due to the insoluble and non-crystalline nature of most membrane peptides and proteins, SSNMR is particularly powerful to investigate their conformations, dynamics, domain assembly, oliogmerization, and the characteristic structural properties in lipid bilayers including insertion orientation and depth, residue-lipid interaction, and membrane per-turbation. Our research is to collect structural and dynamic information and correlate it with biological functions to elucidate the structure-bioactivity relation. In my PhD pro-jects, we have successfully applied various SSNMR techniques to study many interesting membrane peptides including the cell-penetrating peptide (CPP), antimicrobial peptides (AMP), antimicrobial oligomer (AMO), gating helix of K+ channel (KvAP) and trans-membrane 1H channel of influenza M2 protein (M2TM). We also developed a novel paramagnetic-ion-membrane bound paramagnetic relaxation enhancement (PRE) method to provide quantitative long-range distance constrain (~20 y) in membrane-active bio-systems and applied the method to obtain high-resolution residue-specific insertion depth of two membrane peptides, penetratin and M2TM. One main category of my research topics is the cationic membrane peptide. On the one hand, phospholipid membranes have highly hydrophobic interiors that cannot accommodate charged species, while on the other hand, cationic peptides need to insert or translocate across the membrane to conduct biological functions. So, we are motivated to uncover the structural basis of the membrane insertion and translocation. With this motivation, we have studied two kinds of cationic bio-macromolecules, including CPP and AMP. We have experimentally proved that all these Arg-rich peptides generally have strong guanidinium-phosphate interaction with the phospholipids. This charge-charge interaction causes headgroup reorientation and allows the peptide to insert. For CPPs, the guanidinium-phosphate ion pair helps to stabilize the unstructured peptide in the membrane-water interface. The observed peptide-water interaction further minimizes the peptide polarity and makes it more membrane-soluble. We find that two representative CPPs, penetratin and TAT, have highly dynamic and plastic conformations, proposed to facilitate the movement within the membrane. In the penetratin study, the one-side Mn2+-bound PRE method has been developed and applied to study the pep-tide-concentration dependent insertion depth and symmetry in the outer and inner leaflets of the POPC/POPG bilayer. Another important kind of cationic membrane peptides is AMP. Taking PG-1 and its charge reduced mutant IB484 as model AMPs, we have stud-ied the antimicrobial mechanism, and for the first time, provided high-resolution struc-tural information to elucidate the bacterial Gram-selectivity. We find that the interaction manifests the manner of peptide insertion in terms of orientation and depth, which in turn determined the antimicrobial ability in gram positive and negative bacterial membranes. The antimicrobial mechanism of a guanidinium-rich AMO, PMX30016, has also been investigated. The finding of drug-concentration dependant lipid 31P CSA change and the fast uniaxial motion in the interfacial membrane region suggest a subtle and combined antimcicrobial mechanism of membrane potential perturbation and in-plane disruption. Another category of my research topics is the transmembrane ion-conductive channel study, including the gating mechanism of a K+ channel (KvAP) and the blocking mechanism of the M2TM 1H channel by the metal ion inhibitor (Cu2+). We have deter-mined the topology of an isolated gating helix (S4) of KvAP and compared the orientation with that of an intact K+ channel, Kv1.2-Kv2.1 paddle chimera. The identical tilted and rotational angles of the S4 helix in the isolated form and intact protein, and the observed interaction suggest the channel gating might be manifested by the pep-tide-lipid interaction rather than the interaction among different helical domains. Finally, we applied PRE techniques to study the Cu2+-inhibited M2TM channel and obtained high resolution Cu2+ binding structure and long-range distance constraints for the binding structure refinement

High Resolution Structural Characterization of Aβ₄₂ Amyloid Fibrils by Magic Angle Spinning NMR

National Institute of Biomedical Imaging and Bioengineering (U.S.) (EB-003151)National Institute of Biomedical Imaging and Bioengineering (U.S.) (EB-001960)National Institute of Biomedical Imaging and Bioengineering (U.S.) (EB-002026

Formulating a heat- and shear-labile drug in an amorphous solid dispersion: Balancing drug degradation and crystallinity.

We seek to further addresss the questions posed by Moseson et al. regarding whether any residual crystal level, size, or characteristic is acceptable in an amorphous solid dispersion (ASD) such that its stability, enhanced dissolution, and increased bioavailability are not compromised. To address this highly relevant question, we study an interesting heat- and shear-labile drug in development, LY3009120. To study the effects of residual crystallinity and degradation in ASDs, we prepared three compositionally identical formulations (57-1, 59-4, and 59-5) using the KinetiSol process under various processing conditions to obtain samples with various levels of crystallinity (2.3%, 0.9%, and 0.1%, respectively) and degradation products (0.74%, 1.97%, and 3.12%, respectively). Samples with less than 1% crystallinity were placed on stability, and we observed no measurable change in the drug's crystallinity, dissolution profile or purity in the 59-4 and 59-5 formulations over four months of storage under closed conditions at 25 °C and 60% humidity. For formulations 57-1, 59-4, and 59-5, bioavailability studies in rats reveal a 44-fold, 55-fold, and 62-fold increase in mean AUC, respectively, compared to the physical mixture. This suggests that the presence of some residual crystals after processing can be acceptable and will not change the properties of the ASD over time

Multi-criteria decision making for identification of unbalanced bidding

Unbalanced bidding is a serious problem in the competitive bidding practices of construction projects. Identification and prevention of unbalanced bidding is an important and complexity task for owners. This paper aims to propose an identification model of unbalanced bidding from multi-criteria decision making (MCDM) perspective. The VIKOR method is employed to detect unbalanced bidding, in which the line items and bidders are considered as criteria and alternatives in MCDM, respectively. And the engineer’s estimated price is chosen as evaluation benchmarking. Then relative distances between engineer’s estimated price and each bidding unit price are calculated to build decision matrix. The weights of factors are determined using entropy weight method. To illustrate the effectiveness of the proposed model, an application example is tested in detecting unbalanced bidding. Finally, the sensitivity analysis about VIKOR method is given. It shows that the presented model would provide a robust decision making support for owner in identifying unbalanced bidding. First published online 18 December 201

Aggregation challenges in the formulation development of multi-dose peptide products

The formulation development of parenteral peptide therapeutics frequently encounters aggregation challenges. In-depth biophysical understanding of the molecule and formulation are required to achieve formulation robustness. Further, unique considerations need to be given for peptide products that require multi-dose as the use of preservatives can promote aggregation while preservative effectiveness can also be impacted by its interaction with the peptide. This presentation will focus on the reversible and irreversible fibril aggregates in peptide formulations. Biophysical characterization of aggregation and formulation will be discussed in detail. Formation of reversible aggregates and the impact of excipients especially preservatives will be discussed. For the development of fibril-prone peptides, analytical challenges, formulation strategies, as well as predictive test for kinetics will also be discussed. In particular, studies on the temperature-dependent fibril nucleation kinetics and its impact on formulation development will be presented. Please click Additional Files below to see the full abstract

Molecular Dynamics of Neutral Polymer Bonding Agent (NPBA) as Revealed by Solid-State NMR Spectroscopy

Neutral polymer bonding agent (NPBA) is one of the most promising polymeric materials, widely used in nitrate ester plasticized polyether (NEPE) propellant as bonding agent. The structure and dynamics of NPBA under different conditions of temperatures and sample processing are comprehensively investigated by solid state NMR (SSNMR). The results indicate that both the main chain and side chain of NPBA are quite rigid below its glass transition temperature (Tg). In contrast, above the Tg, the main chain remains relatively immobilized, while the side chains become highly flexible, which presumably weakens the interaction between bonding agent and the binder or oxidant fillers and in turn destabilizes the high modulus layer formed around the oxidant fillers. In addition, no obvious variation is found for the microstructure of NPBA upon aging treatment or soaking with acetone. These experimental results provide useful insights for understanding the structural properties of NPBA and its interaction with other constituents of solid composite propellants under different processing and working conditions.National Natural Science Foundation (China) (21120102038)National Natural Science Foundation (China) (21373265)National Natural Science Foundation (China) (21003154

Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5–0.3 ppm for [superscript 13]C line widths and <0.5 ppm [superscript 15]N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via [superscript 1]H detected MAS NMR.National Institutes of Health (U.S.) (Grant EB001960)National Institutes of Health (U.S.) (Grant EB002026

Magic Angle Spinning Nuclear Magnetic Resonance Characterization of Voltage-Dependent Anion Channel Gating in Two-Dimensional Lipid Crystalline Bilayers

National Institutes of Health (U.S.) (EB001960)National Institutes of Health (EB002026

Machine Learning Based Crop Drought Mapping System by UAV Remote Sensing RGB Imagery

Water stress has adverse effects on crop growth and yield, where its monitoring plays a vital role in precision crop management. This paper aims at initially exploiting the potentials of UAV aerial RGB image in crop water stress assessment by developing a simple but effective supervised learning system. Various techniques are seamlessly integrated into the system including vegetation segmentation, feature engineering, Bayesian optimization and Support Vector Machine (SVM) classifier. In particular, wheat pixels are first segmented from soil background by using the classical vegetation index thresholding. Rather than performing pixel-wise classification, pixel squares of appropriate dimension are defined as samples, from which various features for pure vegetation pixels are extracted including spectral and color index (CI) features. SVM with Bayesian optimization is adopted as the classifier. To validate the developed system, a Unmanned Aerial Vehicle (UAV) survey is performed to collect high-resolution atop canopy RGB imageries by using DJI S1000 for the experimental wheat fields of Gucheng town, Heibei Province, China. Two levels of soil moisture were designed after seedling establishment for wheat plots by using intelligent irrigation and rain shelter, where field measurements were to obtain ground soil water ratio for each wheat plot. Comparative experiments by three-fold cross-validation demonstrate that pixel-wise classification, with a high computation load, can only achieve an accuracy of 82.8% with poor F1 score of 71.7%; however, the developed system can achieve an accuracy of 89.9% with F1 score of 87.7% by using only spectral intensities, and the accuracy can be further improved to 92.8% with F1 score of 91.5% by fusing both spectral intensities and CI features. Future work is focused on incorporating more spectral information and advanced feature extraction algorithms to further improve the performance

Enhanced Osseointegration of Hierarchically Structured Ti Implant with Electrically Bioactive SnO₂-TiO₂ Bilayered Surface

The poor osseointegration of Ti implant significantly compromise its application in load-bearing bone repair and replacement. Electrically bioactive coating inspirited from heterojunction on Ti implant can benefit osseointegration but cannot avoid the stress shielding effect between bone and implant. To resolve this conflict, hierarchically structured Ti implant with electrically bioactive SnO2–TiO2 bilayered surface has been developed to enhance osseointegration. Benefiting from the electric cue offered by the built-in electrical field of SnO2–TiO2 heterojunction and the topographic cue provided by the hierarchical surface structure to bone regeneration, the osteoblastic function of basic multicellular units around the implant is significantly improved. Because the individual TiO2 or SnO2 coating with uniform surface exhibits no electrical bioactivity, the effects of electric and topographic cues to osseointegration have been decoupled via the analysis of in vivo performance for the placed Ti implant with different surfaces. The developed Ti implant shows significantly improved osseointegration with excellent bone–implant contact, improved mineralization of extracellular matrix, and increased push-out force. These results suggest that the synergistic strategy of combing electrical bioactivity with hierarchical surface structure provides a new platform for developing advanced endosseous implants