The A beta peptide contributes to Alzheimer's disease through various mechanisms, including cell membrane disruption. While the fibrillar structure of A beta(1-42) in aqueous medium has been elucidated, its oligomer structure remains elusive. We have combined Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), solid-state NMR (ssNMR), and molecular dynamics (MD) approaches to achieve a structural model for A beta(1-42) octamer in lipid bilayers. FTIR data identify conformational transitions of A beta(1-42) to a stable beta-sheet structure. ssNMR analysis allows assignment of 38 out of 42 A beta(1-42) residues, with three additional inter-residue contacts to define the tertiary fold. Combined, MD simulations produce a structural model of A beta(1-42) octamers in a novel sushi-roll fold of in-register cross-beta motif with a lipid-filled internal cavity. The membrane-embedded structure of A beta(1-42) and the mode of peptide-lipid interactions provide a better understanding of A beta neurotoxicity.

Infection with human T-cell leukemia virus type 1 (HTLV-1) can result in adult T-cell leukemia/lymphoma and HTLV-1 associated-myelopathy/tropical spastic paraparesis. The Gag polyprotein - the major structural protein - is crucial for driving virus particle assembly, with the capsid (CA) domain as the key determinant for Gag multimerization. Here, we characterize the immature CA lattice from immature virus particles by using cryo-electron microscopy and tomography (cryo-EM/ET). We report resolving the immature CA lattice to 3.4 Å resolution by single particle analysis (SPA). Our reconstruction reveals that the lattice is stabilized through a trimeric NTD inter-hexamer interface and a dimeric CTD inter-hexamer interface. Further analysis by cryo-ET reveals clear heterogeneity, notably the varying lattice curvatures and the varying distances from the CA layer to the membrane. Intriguingly, inositol hexakisphosphate (IP6) is dispensable for HTLV-1 immature particle assembly and proper immature lattice formation. These observations provide deeper insights into the molecular basis of HTLV-1 immature particle morphology as well as aid in revealing therapeutic targets.

Myocilin-associated glaucoma is a protein-conformational disorder associated with formation of a toxic amyloid-like aggregate. Numerous destabilizing single point variants, distributed across the myocilin olfactomedin β-propeller (OLF, myocilin residues 245-504, 30 kDa) are associated with accelerated disease progression. In vitro, wild type (WT) OLF can be promoted to form thioflavin T (ThT)-positive fibrils under mildly destabilizing (37°C, pH 7.2) conditions. Consistent with the notion that only a small number of residues within a protein are responsible for amyloid formation, 3D 13C-13C solid-state NMR spectra show that OLF fibrils are likely to be composed of only about one third of the overall sequence. Here, we probe the residue composition of fibrils formed de novo from purified full-length OLF. We were able to make sequential assignments consistent with the sequence S331-G-S-L334. This sequence appears once within a previously identified amyloid-prone region (P1, G326AVVYSGSLYFQ) internal to OLF. Since nearly half of the pairs of adjacent residues (di-peptides) in OLF occur only once in the primary structure and almost all the 3-residue sequences (tri-peptides) are unique, remarkably few sequential assignments are necessary to uniquely identify specific regions of the amyloid core. This assignment approach could be applied to other systems to expand our molecular comprehension of how folded proteins undergo fibrillization.

In the maturation process, the immature spherical retroviral capsids reorganize into polymorphic mature capsids. The polymorphism of mature retroviral capsids is caused by insertion of twelve pentameric capsid proteins (CAs) into the hexameric lattice. However, it is still unclear how the same CA switches its conformation to form either pentamer or hexamer assembly that determines the capsid morphology. Here we report our solid state NMR (ssNMR) and molecular dynamics (MD) simulations studies of the assembly of the 237-residue Rous sarcoma virus (RSV) CA, a widely used retroviral template. Torsion angles and dynamics are derived from chemical shift assignments of 224 residues in the RSV CA T=1 capsid assembly, formed entirely by CA pentamers. by comparing with our prior work of the same protein in its tubular assembly comprising exclusively CA hexamers, we reveal, for the first time, site-specific structural differences of pentamer vs hexamer assembly in its native state. Combining our ssNMR constraints with prior published cryoEM structure of T=1 capsid, our model unambiguously proves that selection of hexamer vs pentamer assembly is controlled by a rigid body rotational motion of two domains of RSV CA around its interdomain flexible linker.

During the maturation step, the capsid proteins (CAs) of a retrovirus assemble into polymorphic capsids, whose acute curvature is largely determined by insertion of 12 pentamers into the hexameric lattice. Despite years of intensive research, it remains elusive how the CA switches its conformation between the quasi-equivalent pentameric and hexameric assemblies to generate the acute curvature in the capsid. Here we report the high-resolution structural model of the RSV CA T=1 capsid. By comparing with our prior model of the RSV CA tubular assembly consisting entirely of hexameric lattices, we identify that a dozen of residues are the key to dictate the incorporation of acute curvatures in the capsid assembly. They undergo large torsion angle changes, which result a 34° rotation of the C-terminal domain relative to its N-terminal domain around the flexible interdomain linker, without substantial changes of either the conformation of individual domains or the assembly contact interfaces.

Protein- and peptide-based proton conductors have been extensively studied because of their important roles in biological processes and established potential for bioelectronic device applications. However, despite much progress, the demonstration of long-range proton transport for such materials has remained relatively rare. Herein, we fabricate, electrically interrogate, and physically characterize films from a reflectin-derived polypeptide. The electrical measurements indicate that device-integrated films exhibit proton conductivities with values of ∼0.4 mS/cm and sustain proton transport over distances of ∼1 mm. The accompanying physical characterization indicates that the polypeptide possesses characteristics analogous to those of the parent protein class and furnishes insight into the relationship between the polypeptide's electrical functionality and structure in the solid state. When considered together, our findings hold significance for the continued development and engineering of not only reflectin-based materials but also other bioinspired proton conductors.

Our introduction of density matrix and operator formalism will suffice the analyses of NMR pulse sequences. In this chapter, we will introduce a new concept called tensor. Its implementation will greatly simplify the analyses of spin dynamics in many more complicated scenarios than that of individual spin angular momentum operators. We will first briefly introduce the Wigner-Eckart theory, which provides a framework to describe the general principles regarding tensors rotation. The Wigner-Eckart theory has many more advanced applications that can make your heads (and mine) spin. But please don't be intimidated. We will limit our discussion to its applications relevant to NMR. Specifically, we will demonstrate the derivation of the effect of Magic Angle Spinning (MAS) on interactions essential to NMR. The tensor representation will lead to a general format for the analysis of MAS NMR experiments by AHT. This chapter will be the foundation for our discussion of the recoupling in Chapter 6 and decoupling in Chapter 7.

Aggregates of α-synuclein contribute to the etiology of Parkinson’s Disease. Protein disulfide isomerase (PDI), a chaperone and oxidoreductase, blocks the aggregation of α-synuclein. An S-nitrosylated form of PDI that cannot function as a chaperone is associated with elevated levels of aggregated α-synuclein and is found in brains afflicted with Parkinson’s Disease. The protective role of PDI in Parkinson’s Disease and other neurodegenerative disorders is linked to its chaperone function, yet the mechanism of neuroprotection remains unclear. Using Thioflavin-T fluorescence and transmission electron microscopy, we show here for the first time that PDI can break down nascent fibrils of α-synuclein. Mature fibrils were not affected by PDI. Another PDI family member, ERp57, could prevent but not reverse α-synuclein aggregation. The disaggregase activity of PDI was effective at a 1:50 molar ratio of PDI:α-synuclein and was blocked by S-nitrosylation. PDI could not reverse the aggregation of malate dehydrogenase, which indicated its disaggregase activity does not operate on all substrates. These findings establish a previously unrecognized disaggregase property of PDI that could underlie its neuroprotective function.

By now, we've explained the correlation between the nuclear spin and macroscopic magnetic magnetization, as discussed in Chapter 1. In this chapter, we will introduce some of the most frequently utilized skills for more advanced quantitative analysis in NMR: the rotating frame transformation, from both classical and quantum mechanical perspective. It helps us visualize the motions of magnetization under an on resonance RF pulse by removing the interference of the precession motion at the Larmor frequency. The resonance offset effect will be manifested by detailed derivations, which will be one of the most common adversaries for many recoupling or decoupling sequences. Another common trick, the tilted frame transformation, will be introduced to simplify our discussion of the motion of magnetization in the presence of resonance offset. Following this, quadrature detection will be introduced. We will end this chapter with some qualitative discussion of relaxation phenomena.

We will cover basic concepts regarding the building blocks of NMR spectrometer and probe. This knowledge will serve as a good foundation for more advanced topics regarding NMR probe design. We will start our discussion with more detailed analysis of NMR sensitivity, with a brief coverage on the basic properties of the coaxial cable. Then, our discussion will focus on the introductions of the basic RLC in series and parallel circuits, with detailed derivation to explain their essential electronic properties. They are the fundamental construction blocks in probe design. We will end our discussion with the demonstration of the balanced circuit, which is widely exploited in current NMR probes.

I hope we now established some qualitative physical concepts and pictures about the fundamentals of NMR after the first three chapters. This chapter will introduce the basics of quantitative analysis of NMR experiments by quantum mechanics. A good background in linear algebra will be very beneficial, even if you have no prior knowledge of quantum mechanics. We will start our discussion with introduction of relevant quantum mechanical concepts and principles necessary for our NMR analysis, with emphasis on the density matrix method. Then density matrix will be applied to explain the concept of coherence, and why normally only -1 coherence is detected in our quadrature detection setup. As an example, the evolution of a system consisting of non-interacting spins will be derived under one of the most famous RF pulse sequences, the Hahn Echo, in the presence of isotropic chemical shift. To account for interactions, we will demonstrate how to break down the dipolar interaction into different coherent components. Subsequently we will introduce the operator formalism, which is based on the density matrix method. The operator formalism will be applied to analyze some basic NMR pulse sequences, including the Stimulated Echo, Solid Echo and adiabatic demagnetization. In this process, the effects of nuclear interaction are manifested by their effect over the evolution of the spin system. In this process, another important concept, the coherence transfer will be demonstrated. It is frequently exploited in various NMR pulse sequences, both in solution and ssNMR. To facilitate the analysis, the transformation into fictitious double and zero quantum space will be introduced. They will be employed to demonstrate the excitation and detection of double quantum coherence by a simple (π2)x−τ−(π2)x sequence. It shares the core principles as other advanced pulse sequences to be discussed in Chapter 6 and 7. To generalize our discussion, the Cogwheel phase cycling will be explained as the optimal strategy for coherence pathway selection. The fictitious spin operators will also be applied to analyze the mechanism of cross polarization (CP), one of the most frequently applied signal enhancement pulse sequence in ssNMR. We will end our discussion with the average Hamiltonian theory (AHT), which will be our main theoretical framework to analyze more complicated pulse sequences in subsequent chapters.

Decoupling of interactions is a common and essential practice in protein NMR experiments. High-resolution 13C/15N spectra require efficient proton decoupling. Proton homonuclear decoupling is also necessary to characterize intrinsic relaxation or for proton detection at high MAS. However, it is more complicated to understand than the recoupling of interactions, due to the strong and homogeneous proton dipolar network. Due to the nature of many-body problem, spin physics of decoupling is still a topic not fully understood. Our goal is to introduce the fundamentals of popular theoretical frameworks. We will start our discussion with a qualitative description to differentiate the effect of inhomogeneous and homogeneous interactions on the spectral resolution and relaxation. Then, the mechanism of Lee-Goldburg (LG) sequence to decouple homonuclear dipolar interaction will be discussed, based on AHT. Quantitative analysis of NMR linewidth will be presented to understand the effect of decoupling by MAS alone. Subsequently, decoupling by strong RF irradiation at low MAS will be analyzed in the absence of strong proton homonuclear dipolar coupling network. In the presence of strong proton coupling network, with simultaneous MAS and RF irradiation, interferences arise, and situations immediately become very challenging for AHT. Analytical solutions can be obtained at commensurate MAS and RF irradiation frequencies. To analyze decoupling phenomena beyond this regime, we introduce the Floquet theory, focusing on the principles of Floquet theory.

Protein disulfide isomerase (PDI) is a ubiquitously expressed member of the thioredoxin family with related but independent oxidoreductase and chaperone properties. As a chaperone, PDI plays a crucial role in preventing the aggregation of misfolded proteins. Notably, PDI is known to inhibit alpha‐synuclein (SYN) fibrillization, a pathological hallmark of Parkinson's disease, but the mechanism by which PDI blocks the formation of SYN fibrils is unknown. Here, we report for the first time that PDI not only prevents SYN aggregation but is able to reverse aggregation both in vitro and in cultured cells. Assays using Thioflavin‐T fluorescence and transmission electron microscopy revealed that PDI is able to break down nascent but not mature fibrils. Furthermore, PDI was able to inhibit and reverse cellular fibrillization in HEK293T cells as assessed by protein‐fragment complementation and non‐reducing SDS‐PAGE. These data suggest a novel disaggregase function for PDI that disrupts the SYN fibrils most commonly found in Parkinson's disease. Current treatments for PD are limited to palliative care, but strategies focused on targeted therapeutic intervention are becoming popular. Thus, our work suggests the disaggregase property of PDI may lend itself to therapeutic development for Parkinson's disease.Support or Funding InformationThis work was supported, in part, by a LIFE at UCF Richard Tucker Gerontology Applied Research Award and a UCF College of Medicine competitive research grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recently, Metal-Organic Frameworks (MOFs) derived carbon-based materials have attract wide interest in electrochemical devices due to their large surface area and favorable conductivity. In this work, instead of using MOFs for direct carbon sources, we employed vanadium metal-organic framework (MIL-101(V)) precursor as both carbon sources and vanadium sources for synthesizing carbon-coated Li3V2(PO4)(3) nanocomposites (LVP@M-101). The electrochemical property of LVP@M-101 has been investigated as cathode electrode at a voltage of 3.0-4.8 vs Lithorn/Li, to compare with Li3V2(PO4)(3) prepared using V2O5. It is shown that the composite material displays a remarkably improved electrochemical stability with a high reversible capacity of 113.1 and 105.8 mA h g(-1) at the rate of 0.5C and 1C after 1000 cycles, together with a superior rate performance at various current densities from 0.1C to 10C. Moreover, we have applied ex-situ PXRD and EPR spectroscopy to investigate the lithiation/delithiation process of LVP@M-101 electrode. Through detailed characterizations and electrochemical tests, we believe that the novel nanocomposites LVP@M-101 retain the two-phase transition nature of Li3V2(PO4)(3) and the enhanced cathodic performance in lithium-ion battery is largely due to its unique structural stability. (C) 2018 Elsevier Ltd. All rights reserved.

The orthoretroviral capsid protein (CA) assembles into polymorphic capsids, whose architecture, assembly, and stability are still being investigated. The N-terminal and C-terminal domains of CA (NTD and CTD, respectively) engage in both homotypic and heterotypic interactions to create the capsid. Hexameric turrets formed by the NTD decorate the majority of the capsid surface. We report nearly complete solid-state NMR (ssNMR) resonance assignments of Rous sarcoma virus (RSV) CA, assembled into hexamer tubes that mimic the authentic capsid. The ssNMR assignments show that, upon assembly, large conformational changes occur in loops connecting helices, as well as the short 3(10) helix initiating the CTD. The interdomain linker becomes statically disordered. Combining constraints from ssNMR and cryo-electron microscopy (cryo-EM), we establish an atomic resolution model of the RSV CA tubular assembly using molecular dynamics flexible fitting (MDFF) simulations. On the basis of comparison of this MDFF model with an earlier-derived crystallographic model for the planar assembly, the induction of curvature into the RSV CA hexamer lattice arises predominantly from reconfiguration of the NTD-CTD and CTD trimer interfaces. The CTD dimer and CTD trimer interfaces are also intrinsically variable. Hence, deformation of the CA hexamer lattice results from the variable displacement of the CTDs that surround each hexameric turret. Pervasive H-bonding is found at all interdomain interfaces, which may contribute to their malleability. Finally, we find helices at the interfaces of HIV and RSV CA assemblies have very different contact angles, which may reflect differences in the capsid assembly pathway for these viruses.

The HIV genome materials are encaged by a proteinaceous shell called the capsid, constructed from similar to 1000-1500 copies of the capsid proteins. Because its stability and integrity are critical to the normal life cycle and infectivity of the virus, the HIV capsid is a promising antiviral drug target. In this paper, we review the studies shaping our understanding of the structure and dynamics of the capsid proteins and various forms of their assemblies, as well as the assembly mechanism.

PAP248-286 is a 39-residue fragment (residues 248 to 286) derived from protease cleavage of prostatic acidic phosphatase in semen. The amyloid fibrils formed in vitro by PAP248-286 can dramatically enhance human immunodeficiency virus (HIV) infection. To our knowledge, we present the first report that the HIV-enhancing potency of fibrils formed by PAP248-286 is morphology dependent. We identified pleomorphic fibrils by transmission electron microscopy in two buffer conditions. Our solid-state NMR data showed that these fibrils consist of molecules in distinct conformations. In agreement with NMR, fluorescence measurements confirmed that they are assembled along different pathways, with distinct molecular structures. Furthermore, our cell-based infectivity tests detected distinct HIV-enhancing potencies for fibrils in distinct morphologies. In addition, our transmission electron microscopy and NMR results showed that semen-derived enhancer of viral infection fibrils formed in sodium bicarbonate buffer remain stable over time, but semen-derived enhancer of viral infection fibrils formed in phosphate buffered saline keep evolving after the initial 7 days incubation period. Given time, most of the assemblies in phosphate buffered saline will turn into elongated thin fibrils. They have similar secondary structure but different packing than thin fibrils formed initially after 7 days incubation.

We show the construction of a novel coarse grain model for simulations of HIV capsid assembly based on four structural models of HIV capsid proteins: isolated hexamer 3H47.pdb, tubular assembly 3J34.pdb, isolated pentamer 3P05.pdb and C-terminus dimer 2KOD.pdb. The data demonstrates the derivation of inter-domain motions from all atom Molecular Dynamics simulations and comparison with the motions derived from the analysis of solution NMR results defined in 2M8L.pdb. Snapshots from a representative Monte Carlo simulation with 128 dimeric subunit proteins based on 3J34.pdb are shown in addition to the quantitative analysis of its assembly pathway. Movies of the assembly process are compiled with snapshots of representative simulations of four structural models. The methods and data in this article were utilized in Qiao et al. (in press) [1] to probe the mechanism of polymorphism and curvature control of HIV capsid assembly.

Background: During the maturation process, HIV capsid proteins self-assemble into polymorphic capsids. The strong polymorphism precludes high resolution structural characterization under in vivo conditions. In spite of the determination of structural models for various in vitro assemblies of HIV capsid proteins, the assembly mechanism is still not well-understood.Methods: We report 3D simulations of HIV capsid proteins by a novel coarse grain model that captures the backbone of the rigid segments in the protein accurately. The effects of protein dynamics on assembly are emulated by a static ensemble of subunits in conformations derived from molecular dynamics simulation.Results: We show that HIV capsid proteins robustly assemble into hexameric lattices in a range of conditions where trimers of dimeric subunits are the dominant oligomeric intermediates. Variations of hexameric lattice curvatures are observed in simulations with subunits of variable inter-domain orientations mimicking the conformation distribution in solution. Simulations with subunits based on pentameric structural models lead to assembly of sharp curved structures resembling the tips of authentic HIV capsids, along a distinct pathway populated by tetramers and pentamers with the characteristic quasi-equivalency of viral capsids.Conclusions: Our results suggest that the polymorphism assembly is triggered by the inter-domain dynamics of HIV capsid proteins in solution. The assembly of highly curved structures arises from proteins in conformation with a highly specific inter-domain orientation. Significance: Our work proposes a mechanism of HIV capsid assembly based on available structural data, which can be readily verified. Our model can be applied to other large biomolecular assemblies. Published by Elsevier B.V.