Assembly of Capsids from Hepatitis B Virus Core Protein Progresses through Highly Populated Intermediates in the Presence and Absence of RNA.

The genetic material of viruses is protected by protein shells that are assembled from a large number of subunits in a process that is efficient and robust. Many of the mechanistic details underpinning efficient assembly of virus capsids are still unknown. The assembly mechanism of hepatitis B capsids has been intensively researched using a truncated core protein lacking the C-terminal domain responsible for binding genomic RNA. To resolve the assembly intermediates of hepatitis B virus (HBV), we studied the formation of nucleocapsids and empty capsids from full-length hepatitis B core proteins, using time-resolved small-angle X-ray scattering. We developed a detailed structural model of the HBV capsid assembly process using a combination of analysis with multivariate curve resolution, structural modeling, and Bayesian ensemble inference. The detailed structural analysis supports an assembly pathway that proceeds through the formation of two highly populated intermediates, a trimer of dimers and a partially closed shell consisting of around 40 dimers. These intermediates are on-path, transient and efficiently convert into fully formed capsids. In the presence of an RNA oligo that binds specifically to the C-terminal domain the assembly proceeds via a similar mechanism to that in the absence of nucleic acids. Comparisons between truncated and full-length HBV capsid proteins reveal that the unstructured C-terminal domain has a significant impact on the assembly process and is required to obtain a more complete mechanistic understanding of HBV capsid formation. These results also illustrate how combining scattering information from different time-points during time-resolved experiments can be utilized to derive a structural model of protein self-assembly pathways.

Small-Angle Scattering as a Structural Probe for Nucleic Acid Nanoparticles (NANPs) in a Dynamic Solution Environment.

Nucleic acid-based technologies are an emerging research focus area for pharmacological and biological studies because they are biocompatible and can be designed to produce a variety of scaffolds at the nanometer scale. The use of nucleic acids (ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA)) as building materials in programming the assemblies and their further functionalization has recently established a new exciting field of RNA and DNA nanotechnology, which have both already produced a variety of different functional nanostructures and nanodevices. It is evident that the resultant architectures require detailed structural and functional characterization and that a variety of technical approaches must be employed to promote the development of the emerging fields. Small-angle X-ray and neutron scattering (SAS) are structural characterization techniques that are well placed to determine the conformation of nucleic acid nanoparticles (NANPs) under varying solution conditions, thus allowing for the optimization of their design. SAS experiments provide information on the overall shapes and particle dimensions of macromolecules and are ideal for following conformational changes of the molecular ensemble as it behaves in solution. In addition, the inherent differences in the neutron scattering of nucleic acids, lipids, and proteins, as well as the different neutron scattering properties of the isotopes of hydrogen, combined with the ability to uniformly label biological macromolecules with deuterium, allow one to characterize the conformations and relative dispositions of the individual components within an assembly of biomolecules. This article will review the application of SAS methods and provide a summary of their successful utilization in the emerging field of NANP technology to date, as well as share our vision on its use in complementing a broad suite of structural characterization tools with some simulated results that have never been shared before.

The Fluidity of Phosphocholine and Maltoside Micelles and the Effect of CHAPS.

The dynamics of phosphocholine and maltoside micelles, detergents frequently used for membrane protein structure determination, were investigated using electron paramagnetic resonance of spin probes doped into the micelles. Specifically, phosphocholines are frequently used detergents in NMR studies, and maltosides are frequently used in x-ray crystallography structure determination. Beyond the structural and electrostatic differences, this study aimed to determine whether there are differences in the local chain dynamics (i.e., fluidity). The nitroxide probe rotational dynamics in longer chain detergents is more restricted than in shorter chain detergents, and maltoside micelles are more restricted than phosphocholine micelles. Furthermore, the micelle microviscosity can be modulated with mixtures, as demonstrated with mixtures of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate with n-dodecylphosphocholine, n-tetradecylphosphocholine, n-decyl-ß-D-maltoside, or n-dodecyl-ß-D-maltoside. These results indicate that observed differences in membrane protein stability in these detergents could be due to fluidity in addition to the already determined structural differences.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling.

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Low- q Bicelles Are Mixed Micelles.

Bicelles are used in many membrane protein studies because they are thought to be more bilayer-like than micelles. We investigated the properties of "isotropic" bicelles by small-angle neutron scattering, small-angle X-ray scattering, fluorescence anisotropy, and molecular dynamics. All data suggest that bicelles with a q value below 1 deviate from the classic bicelle that contains lipids in the core and detergent in the rim. Thus not all isotropic bicelles are bilayer-like.

Solution Structure of an Intramembrane Aspartyl Protease via Small Angle Neutron Scattering.

Intramembrane aspartyl proteases (IAPs) comprise one of four families of integral membrane proteases that hydrolyze substrates within the hydrophobic lipid bilayer. IAPs include signal peptide peptidase, which processes remnant signal peptides from nascent polypeptides in the endoplasmic reticulum, and presenilin, the catalytic component of the γ-secretase complex that processes Notch and amyloid precursor protein. Despite their broad biomedical reach, basic structure-function relationships of IAPs remain active areas of research. Characterization of membrane-bound proteins is notoriously challenging due to their inherently hydrophobic character. For IAPs, oligomerization state in solution is one outstanding question, with previous proposals for monomer, dimer, tetramer, and octamer. Here we used small angle neutron scattering (SANS) to characterize n-dodecyl-ß-D-maltopyranoside (DDM) detergent solutions containing and absent a microbial IAP ortholog. A unique feature of SANS is the ability to modulate the solvent composition to mask all but the enzyme of interest. The signal from the IAP was enhanced by deuteration and, uniquely, scattering from DDM and buffers were matched by the use of both tail-deuterated DDM and D2O. The radius of gyration calculated for IAP and the corresponding ab initio consensus model are consistent with a monomer. The model is slightly smaller than the crystallographic IAP monomer, suggesting a more compact protein in solution compared with the crystal lattice. Our study provides direct insight into the oligomeric state of purified IAP in surfactant solution, and demonstrates the utility of fully contrast-matching the detergent in SANS to characterize other intramembrane proteases and their membrane-bound substrates.

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog.

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His-Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr-Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aß42 peptide (Ala-Thr) and the other to the non-pathogenic Aß48 (Thr-Leu). For the former site, we observed more favorable kinetics in lipid bilayer-mimicking bicelles than in detergent solution, indicating that substrate-lipid and substrate-enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions.

Structural investigation of cellobiose dehydrogenase IIA: Insights from small angle scattering into intra- and intermolecular electron transfer mechanisms.

BACKGROUND: Cellobiose dehydrogenases have gained interest due to their potential applications in sectors from biofuel production to biomedical devices. The CDHIIA variant is comprised of a cytochrome domain (CYT), a dehydrogenase domain (DH), and a carbohydrate-binding module (CBM) that are connected by two flexible linkers. Upon cellobiose oxidation at the DH, intramolecular electron transfer (IaET) occurs from the DH to the CYT. In vivo, CDHIIA CYT subsequently performs intermolecular electron transfer (IeET) to a lytic polysaccharide monooxygenase (LPMO). The relevant solution-state CDH domain conformations for IaET and IeET have not been fully characterized. METHODS: Small-angle X-ray and neutron scattering measurements of oxidized CDHIIA from Myriococcum thermophilum and Neurospora crassa were performed to investigate the structural landscape explored in solution by MtCDHIIA and NcCDHIIA in response to cations, pH, and the presence of an electron acceptor, LPMO9D from N. crassa. RESULTS: The scattering data complemented by modeling show that, under oxidizing conditions, MtCDHIIA undergoes global conformational rearrangement in the presence of Ca2+. Oxidized NcCDHIIA exhibits conformational changes upon pH variation and, in the presence of NcLPMO9D, primarily adopts a compact conformation. CONCLUSIONS: These results demonstrate different conformational responses of oxidized MtCDHIIA and NcCDHIIA to changes in environment. The results also reveal a shift in the oxidized NcCDHIIA conformational landscape toward interdomain compaction upon co-incubation with NcLPMO9D. GENERAL SIGNIFICANCE: The present study is the first report on the structural landscapes explored in solution by oxidized cellobiose dehydrogenases under various cation concentrations, pH conditions and in the presence of an electron-accepting LPMO.

Designing Mixed Detergent Micelles for Uniform Neutron Contrast.

Micelle-forming detergents provide an amphipathic environment that mimics lipid bilayers and are important tools used to solubilize and stabilize membrane proteins in solution for in vitro structural investigations. Small-angle neutron scattering (SANS) at the neutron contrast match point of detergent molecules allows observing the signal from membrane proteins unobstructed by contributions from the detergent. However, we show that even for a perfectly average-contrast matched detergent there arises significant core-shell scattering from the contrast difference between aliphatic detergent tails and hydrophilic head groups. This residual signal interferes with interpreting structural data of membrane proteins. This complication is often made worse by the presence of excess empty (protein-free) micelles. We present an approach for the rational design of mixed micelles containing a deuterated detergent analog, which eliminates neutron contrast between core and shell and allows the micelle scattering to be fully contrast-matched to unambiguously resolve membrane protein structure using solution SANS.

Tuning micelle dimensions and properties with binary surfactant mixtures.

Detergent micelles are used in many areas of research and technology, in particular, as mimics of the cellular membranes in the purification and biochemical and structural characterization of membrane proteins. Applications of detergent micelles are often hindered by the limited set of properties of commercially available detergents. Mixtures of micelle-forming detergents provide a means to systematically obtain additional micellar properties and expand the repertoire of micelle features available; however, our understanding of the properties of detergent mixtures is still limited. In this study, the shape and size of binary mixtures of seven different detergents commonly used in molecular host-guest systems and membrane protein research were investigated. The data suggests that the detergents form ideally mixed micelles with sizes and shapes different from those of pure individual micelles. For most measurements of size, the mixtures varied linearly with detergent mole fraction and therefore can be calculated from the values of the pure detergents. We propose that properties such as the geometry, size, and surface charge can be systematically and predictably tuned for specific applications.

Dependence of micelle size and shape on detergent alkyl chain length and head group.

Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2-1.5 Šper carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ∼16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.

Dehaloperoxidase-hemoglobin from Amphitrite ornata is primarily a monomer in solution.

The crystal structures of the dehaloperoxidase-hemoglobin from A. ornata (DHP A) each report a crystallographic dimer in the unit cell. Yet, the largest dimer interface observed is 450 Å(2), an area significantly smaller than the typical value of 1200-2000 Å(2) and in contrast to the extensive interface region of other known dimeric hemoglobins. To examine the oligomerization state of DHP A in solution, we used gel permeation by fast protein liquid chromatography and small-angle X-ray scattering (SAXS). Gel permeation experiments demonstrate that DHP A elutes as a monomer (15.5 kDa) and can be separated from green fluorescent protein, which has a molar mass of 27 kDa, near the 31 kDa expected for the DHP A dimer. By SAXS, we found that DHP A is primarily monomeric in solution, but with a detectable level of dimer (~10%), under all conditions studied up to a protein concentration of 3.0 mM. These concentrations are likely 10-100-fold lower than the K(d) for dimer formation. Additionally, there was no significant effect either on the overall conformation of DHP A or its monomer-dimer equilibrium upon addition of the DHP A inhibitor, 4-iodophenol.