Size Exclusion Chromatography Coupled With Multiangle Light Scattering Research Articles

Lipid composition affects the thermal stability of cytochrome P450 3A4 in nanodiscs

Nanodiscs (NDs), self-assembled lipid bilayers encircled by membrane scaffold proteins (MSPs), offer a versatile platform for the reconstitution of membrane proteins for structural and biochemical investigations. Saturated, isoprenoid lipids are commonly found in thermophiles and have been associated with thermotolerance. To test whether these lipids confer additional stability on ND-incorporated membrane proteins, this study focuses on the thermal stability of human cytochrome P450 3A4 (CYP3A4) inside NDs composed of different phosphocholine lipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). NDs were characterized using size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and densitometric SDS-PAGE. CYP3A4-DPhPC-NDs were found to comprise three MSP copies instead of the canonical dimer, as reported before for the empty NDs. Rapid, thermally induced unfolding of CYP3A4 inside NDs measured using circular dichroism and differential scanning fluorimetry (nanoDSF) revealed that the CYP3A4 melting temperature was dependent on ND composition. In POPC and DMPC-CYP3A4-NDs the melting temperature was comparable to CYP3A4 without NDs (59 °C). CYP3A4 in DPhPC-NDs showed an increase in melting temperature of 4 °C. Decline in CYP3A4 integrity as well as ND aggregation and disintegration occur at similar rates for all membrane types when subjected to exposure at 37 °C for several hours. The POPC and DMPC- CYP3A4-NDs show significant lipid loss over time, which is not observed for DPhPC-NDs. The results demonstrate that thermally induced denaturation of protein-NDs is a complex, multifaceted process, which is not represented well by rapid thermal unfolding experiments.

Preparation of Composite Films of Sodium Alginate-Based Extracted from Seaweeds Macrocystis pyrifera and Lessonia trabeculata Loaded with Aminoethoxyvinylglycine

Composite films are an alternative in the replacement of synthetic polymers. These films can be prepared from polysaccharides and used to store various drugs to be applied in different areas. In addition, aminoethoxyvinylglycine (AVG) is an ethylene inhibitor that prolongs shelf life of food. For this reason, this research aimed to take advantage of the brown algae Macrocystis pyrifera and Lessonia trabeculata to extract sodium alginate and produce films composed of sodium alginate/ kappa-carrageenan/iota-carrageenan, which were plasticized with glycerol and polyethylene glycol 400 and loaded with aminoethoxyvinylglycine. The extracted sodium alginate was characterized by 1 H and 13C nuclear magnetic resonance (NMR), size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), thermogravimetry (TG/DTG), Fourier transform infrared (FTIR), and X-ray diffraction (XRD); and the composite films were characterized by FTIR, XRD, TG/DTG, and scanning electron microscopy (SEM). Then, the drug release kinetics were investigated using Higuchi and Korsmeyer-Peppas kinetic models. The extracted alginates obtained were of low molecular weight, and the films showed desirable properties for AVG release. Furthermore, drug release profiles revealed that AVG release is governed by Fick’s Law, and this is favored at low temperatures. In summary, sodium alginate allows the preparation of composite films, which can replace synthetic polymers to be used in the loading and releasing of drugs.

Determination of the Absolute Molar Mass of [Fe-S]-Containing Proteins Using Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS).

Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) is a technique that determines the absolute molar mass (molecular weight) of macromolecules in solution, such as proteins or polymers, by detecting their light scattering intensity. Because SEC-MALS does not rely on the assumption of the globular state of the analyte and the calibration of standards, the molar mass can be obtained for proteins of any shape, as well as for intrinsically disordered proteins and aggregates. Yet, corrections need to be made for samples that absorb light at the wavelength of the MALS laser, such as iron–sulfur [Fe-S] cluster-containing proteins. We analyze several examples of [2Fe-2S] and [4Fe-4S] cluster-containing proteins, for which various corrections were applied to determine the absolute molar mass of both the apo- and holo-forms. Importantly, the determination of the absolute molar mass of the [2Fe-2S]-containing holo-NEET proteins allowed us to ascertain a change in the oligomerization state upon cluster binding and, thus, to highlight one essential function of the cluster.

Isolated Heme A Synthase from Aquifex aeolicus Is a Trimer.

The integral membrane protein heme A synthase (HAS) catalyzes the biosynthesis of heme A, which is a prerequisite for cellular respiration in a wide range of aerobic organisms. Previous studies have revealed that HAS can form homo-oligomeric complexes, and this oligomerization appears to be evolutionarily conserved among prokaryotes and eukaryotes and is shown to be essential for the biological function of eukaryotic HAS. Despite its importance, little is known about the detailed structural properties of HAS oligomers. Here, we aimed to address this critical issue by analyzing the oligomeric state of HAS from Aquifex aeolicus (AaHAS) using a combination of techniques, including size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), cross-linking, laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS), and single-particle electron cryomicroscopy (cryo-EM). Our results show that HAS forms a thermostable trimeric complex. A cryo-EM density map provides information on the oligomerization interface of the AaHAS trimer. These results provide structural insights into HAS multimerization and expand our knowledge of this important enzyme.IMPORTANCE Heme A is a vital redox cofactor unique for the terminal cytochrome c oxidase in mitochondria and many microorganisms. It plays a key role in oxygen reduction by serving as an electron carrier and as the oxygen-binding site. Heme A is synthesized from heme O by an integral membrane protein, heme A synthase (HAS). Defects in HAS impair cellular respiration and have been linked to various human diseases, e.g., fatal infantile hypertrophic cardiomyopathy and Leigh syndrome. HAS exists as a stable oligomeric complex, and studies have shown that oligomerization of eukaryotic HAS is necessary for its proper function. However, the molecular architecture of the HAS oligomeric complex has remained uncharacterized. The present study shows that HAS forms trimers and reveals how the oligomeric arrangement contributes to the complex stability and flexibility, enabling HAS to perform its catalytic function effectively. This work provides the basic understanding for future studies on heme A biosynthesis.

Nucleotide-Dependent Dimerization and Conformational Switching of Atlastin.

A common feature of dynamin-related proteins (DRPs) is their use of guanosine triphosphate (GTP) to control protein dynamics. In the case of the endoplasmic- reticulum- (ER)-resident membrane protein atlastin (ATL), GTP binding and hydrolysis result in membrane fusion of ER tubules and the generation of a branched ER network. In this chapter, we describe two independent methods for dissecting the mechanism underlying nucleotide-dependent quaternary structure and conformational changes of ATL, focusing on size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and Förster resonance energy transfer (FRET), respectively. The high temporal resolution of the FRET-based assays enables the ordering of the molecular events identified in structural and equilibrium-based SEC-MALS studies. In combination, these complementary methods report on the oligomeric states of a system at equilibrium and timing of key steps along the enzyme's catalytic cycle. These methods are broadly applicable to proteins that undergo ligand-induced dimerization and/or conformational changes.

Biotechnological Production of Poly(3-Hydroxybutyrate-co-4-Hydroxybutyrate-co-3-Hydroxyvalerate) Terpolymer by Cupriavidus sp. DSM 19379.

The terpolymer of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 4-hydroxybutyrate (4HB) was produced employing Cupriavidus sp. DSM 19379. Growth in the presence of γ-butyrolactone, ε-caprolactone, 1,4-butanediol, and 1,6-hexanediol resulted in the synthesis of a polymer consisting of 3HB and 4HB monomers. Single and two-stage terpolymer production strategies were utilized to incorporate the 3HV subunit into the polymer structure. At the single-stage cultivation mode, γ-butyrolactone or 1,4-butanediol served as the primary substrate and propionic and valeric acid as the precursor of 3HV. In the two-stage production, glycerol was used in the growth phase, and precursors for the formation of the terpolymer in combination with the nitrogen limitation in the medium were used in the second phase. The aim of this work was to maximize the Polyhydroxyalkanoates (PHA) yields with a high proportion of 3HV and 4HB using different culture strategies. The obtained polymers contained 0–29 mol% of 3HV and 16–32 mol% of 4HB. Selected polymers were subjected to a material properties analysis such as differential scanning calorimetry (DSC), thermogravimetry, and size exclusion chromatography coupled with multi angle light scattering (SEC-MALS) for determination of the molecular weight. The number of polymers in the biomass, as well as the monomer composition of the polymer were determined by gas chromatography.

The ICP27 Homology Domain of the Human Cytomegalovirus Protein UL69 Adopts a Dimer-of-Dimers Structure.

ABSTRACTThe UL69 protein from human cytomegalovirus (HCMV) is a multifunctional regulatory protein and a member of the ICP27 protein family conserved throughout herpesviruses. UL69 plays many roles during productive infection, including the regulation of viral gene expression, nuclear export of intronless viral RNAs, and control of host cell cycle progression. Throughout the ICP27 protein family, an ability to self-associate is correlated with the functions of these proteins in transactivating certain viral genes. Here, we determined the domain boundaries of a globular ICP27 homology domain of UL69, which mediates self-association, and characterized the oligomeric state of the isolated domain. Size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) revealed that residues 200 to 540 form a stable homo-tetramer, whereas a shorter region comprising residues 248 to 536 forms a homo-dimer. Structural analysis of the UL69 tetramer by transmission electron microscopy (TEM) revealed a dimer-of-dimers three-dimensional envelope with bridge features likely from a region of the protein unique to betaherpesviruses. The data provide a structural template for tetramerization and improve our understanding of the structural diversity and features necessary for self-association within UL69 and the ICP27 family.

Highly Pure Soluble Chimeras of the Intracellular Domain of Anionic Pentameric Ligand-Gated Ion Channels

The past few years have seen remarkable advances in structural information gleaned from atomic resolution X-ray crystallographs of important anionic and cationic pentameric ligand-gated ion channels (pLGIC) from the Cys-loop superfamily. The highly conserved extracellular domain (ECD) and transmembrane domain (TMD), and the diverse intracellular domain (ICD) constitute each subunit of eukaryotic pLGICs. The ECD and TMD of different Cys-loop receptors are prime targets for several clinical drugs. These drugs are thus notorious for binding indiscriminately to undesired receptors within the superfamily resulting in a wide range of unwanted side-effects. Intriguingly, the marked variability of ICDs between different subtypes can be exploited to aid in the development of newer drugs with refined specific binding. However, the ICD of any Cys-loop receptor has yet to be fully characterized. The two prominent anion-conducting pLGICs, glycine-α1 (Gly-α1) and γ-amino butyric acid type A-ρ1 (GABAA-ρ1) receptors, of the Cys-loop superfamily are critical in mediating postsynaptic inhibition in the central nervous system. Dysfunction of these pLGICs has been implicated in numerous serious neuropsychiatric and neurological diseases. The goal of our current project was to develop a strategy for protein expression and purification of the ICD of Gly-α1 as well as GABAA-ρ1 receptors. Here, we generated chimeras containing maltose-binding protein (MBP) fused to the N-terminus of the ICD of either Gly-α1 receptor or that of GABAA-ρ1 receptor and separated by a short alanine linker. We expressed these chimeras in E. coli and were able to purify both proteins of interest to homogeneity by employing 3-step purification. Further characterization to determine size using size exclusion chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) revealed the oligomeric state. Experiments are underway to gain detailed structural insights.

NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma-tubulin complex.

Mitotic‐spindle organizing protein associated with a ring of γ‐tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ‐tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC‐MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha‐helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N‐terminus (residues 1–250) of GCP3 (γ‐tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.

EFFECT OF MASTICATION ON THE STRUCTURE OF MICROGEL PRESENT IN NATURAL RUBBER

ABSTRACT Mechanical or thermal mastication experiments were performed on three commercial natural rubber (NR) samples of TSR10 grade made from latex of three different clones (GT1, PB235, and RRIM600). The mesostructure (different gel or aggregate fractions, structure of random coils of cis-1,4-polyisoprene) of all the NR samples was fully characterized by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), using pretreated SEC columns. This method was used to quantify and investigate the structure of the little-studied smaller microaggregates, constituting the microgel fraction smaller than 1 μm (Microgel<1μ) of NR. The three unmasticated NR samples showed no difference in terms of microaggregate structure. Conversely, microaggregates appeared denser after mastication. This phenomenon was found to depend on the mastication conditions, as mechanically masticated NR samples had smaller (lower radius of gyration) and more compact microaggregates than thermally masticated samples. Macrogel also behaved differently depending on the mastication conditions. Mechanical mastication conditions allowed a higher degradation of the macrogel compared with thermal mastication conditions.

Molecular characterization of methanogenic N5-methyl-tetrahydromethanopterin: Coenzyme M methyltransferase

Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na+ across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS.

Application of recent advances in hydrodynamic methods for characterising mucins in solution.

Mucins are the primary macromolecular component of mucus--nature's natural lubricant--although they are poorly characterised heterogeneous substances. Recent advances in hydrodynamic methodology now offer the opportunity for gaining a better understanding of their solution properties. In this study a combination of such methods was used to provide increased understanding of a preparation of porcine intestinal mucin (PIM), MUC2 mucin, in terms of both heterogeneity and quantification of conformational flexibility. The new sedimentation equilibrium algorithm SEDFIT-MSTAR is applied to yield a weight average (over the whole distribution) molar mass of 7.1 × 10(6) g mol(-1), in complete agreement with size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), which yielded a value of 7.2 × 10(6) g mol(-1). Sedimentation velocity profiles show mucin to be very polydisperse, with a broad molar mass distribution obtained using the Extended Fujita algorithm, consistent with the elution profiles from SEC-MALS. On-line differential pressure viscometry coupled to the SEC-MALS was used to obtain the intrinsic viscosity [η] as a function of molar mass. These data combined with sedimentation coefficient data into the global conformation algorithm HYDFIT show that PIM has a flexible linear structure, with persistence length L p ~10 nm and mass per unit length, M L ~2380 g mol(-1) nm(-1), consistent with a Wales-van Holde ratio of ~1.2 obtained from the concentration dependence of the sedimentation coefficient.

Characterization of chain dimensions of poly(ε-caprolactone) diols in THF by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS)

In this work, we studied the chain dimensions, shape, and thermodynamic characteristics of poly(e-caprolactone) diols (HO-PCL-OH) in solution. PCL diol samples of different molecular weights and architectures were synthesized using immobilized Yarrowia lipolytica lipase as catalyst. Gel permeation chromatography (GPC) with online right-angle laser-light scattering (RALLS), differential viscometer (DV), and interferometric refractometer IRS detectors offered a proper way to obtain information on thermodynamic characteristics and chain flexibility. The weight-average molecular weights of the PCL diol samples (M w ) ranged from 2,750 to 13,120 uma. The z-average radius of hydrodynamic volume (<R h 2 > z 1/2 ) vs. z-average molecular weight curve (M z ) could be satisfactorily fitted to a power-law equation. Mark–Houwink–Sakurada parameters (K = 2.74 × 10−3 dL/g and a = 0.64) were derived from the plots of [η] w against M w . The overall results clearly suggest that a flexible geometry is present in tetrahydrofuran (THF) solution of PCL at 33 °C. Unlike other PCL systems, PCL diols are more hydrophilic and expected to form associated species. They behave differently from common hydrophobic polymers and do not strictly conform to thermodynamic relationships generally used in Polymer Science. A more compact geometry is present for Mn lower than 4,000 Da. For higher molecular weights, the chain expands and become more elongated. Plots of ln [η] w against ln M w (MHS equation) and of log R hz against log M z suggest that PCL diols behave as flexible chains in a good solvent.

Physicochemical study by multi-angle light scattering of water-soluble methacryloylaminophenylarsonate copolymers

The properties of aqueous solutions of polyelectrolytes of sodium o- and p-methacryloylaminophenylarsonate (o- and p- MAPHA-Na) with acrylamide (AAD) or sodium methacrylate (MA-Na): poly(sodium o-methacryloylaminophenylarsonate) (poly(o-MAPHA-Na)), poly(sodium o-methacryloylaminophenylarsonate-co-acrylamide) (poly(o-MAPHA-Na-co-AAD)) (26:74), poly(sodium o-methacryloylaminophenylarsonate-co-sodium methacrylate) (poly(o- MAPHA-Na-co-AM-Na) (35:65) and (14:86), and poly(sodium p-methacryloylaminophenylarsonate-co-sodium methacrylate) (poly(p- MAPHA-Na-co-AM-Na)) (34:66) were studied by off-line, multi-angle light scattering, and in-line using size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and refractometric (RI) detection. The Mw value corresponding to the homopolymer poly(o-MAPHA-Na) was 1,300,000 g/mol and for the copolymers the Mw and gyration radius values were decreased when the AAD or AM-Na comonomer composition increased. The macromolecular parameters obtained using SEC-MALS were in agreement with the values measured in the off-line mode. Also, SEC-MALS experiments allowed examination of the radius of gyration-Mw relationship in order to determine the polyelectrolyte conformation (ρ) in solution; these values fell between 0.7-0.53, indicating that the polyelectrolytes are random coils at θ-conditions. In addition, the effect of the pH on the macromolecular parameters was evaluated. At pH 4.5, the Mw and gyration radius values corresponding to poly(o-MAPHA-Na) and poly(o-MAPHA-Na-co-AAD) (26/74) remained very close to those measured at pH 7. However, <r g 2 > values for poly(o-MAPHA-Na-co-AM-Na) copolymers at pH 4.5 were less than pH 7, indicating that the presence of AM-Na reduced the size of the copolymer through intermolecular interactions.

Evidences of Long‐Chain Branching in Ziegler‐Natta Polyethylene Homopolymers as Studied via SEC‐MALS and Rheology

SummaryComprehensive polymer architectural studies were carried out on a group of Ziegler‐Natta (Z‐N) polyethylene homopolymers via size‐exclusion chromatography coupled with multi‐angle light scattering (SEC‐MALS) and rheology. Although Z‐N resins are traditionally viewed as linear polymers, both SEC‐MALS and rheology results strongly indicate that there are topological variations, presumably long‐chain branching (LCB) or possibly hyper‐branches, in this group of supposedly linear Z‐N homopolymers. These Z‐N homopolymers exhibit very complex elution behavior that resulted in anomalous elution profiles in SEC. It appeared that some high molecular weight (MW) and long‐chain branched components followed a non‐size exclusion separation mechanism on SEC columns, co‐eluting with low MW and linear polyethylene components, causing a delayed elution. The possible origin of LCB in these Z‐N polymers is also discussed.

Characterization of gelatine and acid soluble collagen by size exclusion chromatography coupled with multi angle light scattering (SEC-MALS).

Acid soluble collagen (ASC) and a cattle hide gelatine were analyzed by size exclusion chromatography (SEC) coupled with multi angle light scattering (MALS). The SEC system was calibrated with ASC and its cyan bromide cleavage products. The accuracy of calibration was confirmed by MALS by measuring the mass-average molar masses (Mw). ASC acted as a mixture of two polymer standards of Mw = 90 and 180 kg/mol, respectively. The elution behavior of the gelatine in SEC-MALS was similar to that of ASC. Therefore, the determination of the molar mass distribution of this gelatine was possible either by SEC, using a calibration curve, or by MALS by direct measurement of Mw. According to the scaling law <s2>(1/2) = KMalpha, alpha = 0.78 was determined for the gelatine. This alpha could reflect a structure in solution, which is more similar to an ellipsoid than to a random coil.

Association Behavior of Polyurethanes in Polar Solvents Studied by SEC Coupled with Light Scattering

The solution behavior of non-carboxylated and carboxylated polyurethanes (PU) in THF, LiBr/DMF, and DMF was studied by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS). This paper focuses on the association of PU macromolecules in DMF considering the influences of composition, solution concentration, and temperature. The degree of association increases with increasing amounts of polar urethane and carboxyl groups, with decreasing solution concentration, and by increasing the temperature of measurements or heat treatment—all of which demonstrate the role of impurities present in DMF on PU dilute solution behavior.

STUDY OF POLYSTYRENE-BLOCK-POLY(METHYLMETHACRYLATE) MICELLES BY SEC/MALS. DETERMINATION OF MOLECULAR WEIGHTS AND SIZE DISTRIBUTION

Polystyrene-block-poly(methyl methacrylate) micelles in 1,4-dioxane/cyclohexane were studied by size exclusion chromatography coupled with multi angle light scattering (SEC/MALS). A sufficient unimer — micelles resolution achieved by SEC made it possible to use MALS detector for the direct determination of distributions and corresponding averages of molecular weights and gyration radii of micelles. Contrary to the standard static light scattering approach,the SEC/MALS technique provided these averages in one analytic run with a minimum amount of copolymer sample. In most systems, very narrow molecular weights and size distributions of micelles were obtained. For a given copolymer, the micellar association number increased with decreasing thermodynamic quality of solvent. Under given thermodynamic conditions, it increased with increasing length of insoluble poly(methyl methacrylate) block and with decreasing length of soluble polystyrene block. The micellar radius of gyration was predominantly controlled by the molecular weight of the copolymer.