Dynamics of Leaf- and Root-Specific Biomarkers during 1-Year of Litter Decomposition

Root-specific and leaf-specific biomarkers have been used for decades to identify the origin of organic materials in soils and sediments. However, quantitative approaches require appropriate knowledge about the fate of these indicator molecules during degradation. To clarify this issue, we performed a 1-year incubation experiment with fine root and leaf material of six temperate tree species: European ash (Fraxinus excelsior), European beech (Fagus sylvatica), Oak spec. (Quercus spec.), Linden spec. (Tilia spec.), Norway spruce (Picea abies) and Scots pine (Pinus sylvatica). Only one molecule, x,16-dihydroxy hexadecanoic acid (x,16-C16), could be validated as a general leaf-specific biomarker for the set of all species. For roots, no general root biomarker was found. Ester-bound tricosanol (C23-OH) could be validated for five out of six species; 20-hydroxy eicosanoic acid (ωC20) could be validated for four out of six species, leaving Norway spruce without a suitable root biomarker. The results of this study suggest that the validity of leaf- and root-derived ester-bound lipids as biomarkers is highly species dependent and does not always coincide with previous findings. Concentrations of root- and leaf-derived ester-bound lipids did not stay constant within 1 year of degradation and changed without a linear trend. The change of concentrations seems to be highly species dependent. This might be due to a different structure and arrangement of the individual monomers in cutin and suberin per species, and, therefore, a different accessibility of bond cleaving enzymes. The usefulness of root and leaf biomarkers is context dependent. Our results suggest that general assumptions about litter input to forest soils solely based on biomarker analysis have to be considered carefully.

A tetrameric assembly of saposin A: increasing structural diversity in lipid transfer proteins

Saposins are lipid transfer proteins required for the degradation of sphingolipids in the lysosome. These small proteins bind lipids by transitioning from a closed, monomeric state to an open conformation exposing a hydrophobic surface that binds and shields hydrophobic lipid tails from the aqueous environment. Saposins form a range of multimeric assemblies to encompass these bound lipids and present them to hydrolases in the lysosome. This lipid-binding property of human saposin A has been exploited to form lipoprotein nanodiscs suitable for structural studies of membrane proteins. Here we present the crystal structure of a unique tetrameric assembly of murine saposin A produced serendipitously, following modifications of published protocols for making lipoprotein nanodiscs. The structure of this new saposin oligomer highlights the diversity of tertiary arrangement that can be adopted by these important lipid transfer proteins.

Investigating the Lipid Selectivity of the Ammonium Transporter AmtB in Heterogeneous Nanodiscs

The structure and function of membrane proteins can be significantly impacted by the surrounding lipid environment, but membrane protein-lipid interactions in lipid bilayers are often difficult to study due to their transient and polydisperse nature. Here, we used two native mass spectrometry (MS) approaches to investigate how the Escherichia coli ammonium transporter (AmtB) selectively remodels its local lipid environment in heterogeneous lipoprotein nanodiscs. First, we used gas-phase ejection to isolate AmtB with bound lipids from heterogeneous nanodiscs with different combinations of lipids. Second, we used solution-phase detergent flash extraction as an orthogonal approach to study AmtB remodeling with native MS. Flash extraction of AmtB showed that Triton X-100 retains lipid selectivity, but C8E4 distorts preferential lipid interactions. Both approaches reveal that AmtB has a few tight binding sites for PC, is selective for binding PG over-all, and is nonselective for PE, providing a detailed picture of how AmtB binds different lipid head groups in the context of mixed lipid bilayers.

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids

Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles. Here, we review the structures and mechanisms of transmembrane targets and their interactions with lipids including phosphoinositides (PIs), as resolved using nanodisc systems and methods including cryo-electron microscopy (cryo-EM) and X-ray diffraction (XRD). We focus on therapeutic targets including several G protein-coupled receptors (GPCRs), as well as ion channels and transporters that are driving the development of next-generation native nanodiscs. The design of new synthetic polymers and complementary biophysical tools bodes well for the future of drug discovery and structural biology of native membrane:protein assemblies (memteins).

Structure and function at the lipid–protein interface of a pentameric ligand-gated ion channel

Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have remained elusive. To some extent, this is because tightly bound lipids are often located at the periphery of proteins, where the quality of density maps is usually poorer, and because they may be outcompeted by detergent molecules used during standard purification procedures. As a step toward characterizing natively bound lipids in the superfamily of pentameric ligand-gated ion channels (pLGICs), we applied single-particle cryogenic electron microscopy to fragments of native membrane obtained in the complete absence of detergent-solubilization steps. Because of the heterogeneous lipid composition of membranes in the secretory pathway of eukaryotic cells, we chose to study a bacterial pLGIC (ELIC) expressed in Escherichia coli’s inner membrane. We obtained a three-dimensional reconstruction of unliganded ELIC (2.5-Å resolution) that shows clear evidence for two types of tightly bound lipid at the protein–bulk-membrane interface. One of them was consistent with a “regular” diacylated phospholipid, in the cytoplasmic leaflet, whereas the other one was consistent with the tetra-acylated structure of cardiolipin, in the periplasmic leaflet. Upon reconstitution in E. coli polar-lipid bilayers, ELIC retained the functional properties characteristic of members of this superfamily, and thus, the fitted atomic model is expected to represent the (long-debated) unliganded-closed, “resting” conformation of this ion channel. Notably, the addition of cardiolipin to phosphatidylcholine membranes restored the ion-channel activity that is largely lost in phosphatidylcholine-only bilayers.

Cryo-EM structure of type 1 IP3R channel in a lipid bilayer

Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is the predominant Ca2+-release channel in neurons. IP3R1 mediates Ca2+ release from the endoplasmic reticulum into the cytosol and thereby is involved in many physiological processes. Here, we present the cryo-EM structures of full-length rat IP3R1 reconstituted in lipid nanodisc and detergent solubilized in the presence of phosphatidylcholine determined in ligand-free, closed states by single-particle electron cryo-microscopy. Notably, both structures exhibit the well-established IP3R1 protein fold and reveal a nearly complete representation of lipids with similar locations of ordered lipids bound to the transmembrane domains. The lipid-bound structures show improved features that enabled us to unambiguously build atomic models of IP3R1 including two membrane associated helices that were not previously resolved in the TM region. Our findings suggest conserved locations of protein-bound lipids among homotetrameric ion channels that are critical for their structural and functional integrity despite the diversity of structural mechanisms for their gating.

Mechanosensitive channel YnaI has lipid-bound extended sensor paddles

The general mechanism of bacterial mechanosensitive channels (MS) has been characterized by extensive studies on a small conductance channel MscS from Escherichia coli (E. coli). However, recent structural studies on the same channel have revealed controversial roles of various channel-bound lipids in channel gating. To better understand bacterial MscS-like channels, it is necessary to characterize homologs other than MscS. Here, we describe the structure of YnaI, one of the closest MscS homologs in E. coli, in its non-conducting state at 3.3 Å resolution determined by cryo electron microscopy. Our structure revealed the intact membrane sensor paddle domain in YnaI, which was stabilized by functionally important residues H43, Q46, Y50 and K93. In the pockets between sensor paddles, there were clear lipid densities that interact strongly with residues Q100 and R120. These lipids were a mixture of natural lipids but may be enriched in cardiolipin and phosphatidylserine. In addition, residues along the ion-conducting pathway and responsible for the heptameric assembly were discussed. Together with biochemical experiments and mutagenesis studies, our results provide strong support for the idea that the pocket lipids are functionally important for mechanosensitive channels.

The relationship between structural lipids of sheep wool with its individual macrostructural components, chemical composition and physical indicators

The data on the peculiarities of the structural organization, chemical composition and physical parameters of sheep wool of different breeds depending on the type of their hair are presented. It has been found that the down fibers of ewes of the Ukrainian Carpathian Mountain breed possess the lowest content of β-keratosis (10.2%) and the highest content of α-keratosis (64.4%). In the fine wool of Ascanian ewes and Prekos ewes, the content of β-keratosis is 12.9 and 11.5%, respectively, and the highest content of it (15.1%) is contained in the guard fibers of the Carpathian Mountain ewes. However, in the down fibers of these ewes and the Prekos breed ewes, there is the highest content of γ-keratosis ― 28.4 and 28.7%, the total sulfur and cystine (2.9 and 2.9 and 11.2 and 11.5%), respectively. Besides that, the guard fibers contain the lowest content of both γ-keratosis (58.2%) and sulfur and cystine (2.7 and 9.0%), respectively. It has been established that different categories of fibers contain different amounts of total lipids. The smallest amounts of free lipids are found in the thin down of the Carpathian Mountain ewes (0.75%), the thin wool of the Prekos ewes (0.71%) and Ascanian ewes (0.83%), and the largest number of them is found in the semi-coarse guard fibers of the Carpathian Mountain sheep (1.39%). For bound lipids, a diametrically opposite difference was established: the largest amount of lipids was found in the thin down (1.85%), and the smallest amount — in the semi-coarse guard fibers (1.47%). In the guard fibers, the biggest amount of free lipids is accounted for the fraction of non-esterified cholesterol (64.9% versus 56.5% in the down, 57.7 in the wool of Ascanian ewes and 63.3% in the Prekos ewes), and the least of all they contain the fraction of non-esterified fatty acids (9.6%), and another sterol fraction (9.2%). The fibers of the Prekos breed sheep are noted with the lowest content of esterified cholesterol (8.9%) and the highest content of non-esterified fatty acids. But the fraction of polar lipids consists of almost 50% of ceramides and sulfolipids (more than 20%). At the same time, ceramides account for no more than 40% in the fraction of bound lipids. Physical indicators of wool to some extent reflect the peculiarities of its structure and chemical composition. Thus, the guard fibers have the highest strength (9.1 cN/tex) and fineness (48.8 μm), which is natural, because the guard has the highest content of β-keratose, i.e. cuticle, and the highest amount of lipids. Instead, the thinnest fibers are down fibers (16.9 μm) and they are the weakest (7.0 cN/tex) and these fibers contain the least β-keratose. Thus, there is a direct relationship between the content of the free lipid fraction and the fiber diameter (r = 0.996; 0.887; 0746 for down, fine and semi-coarse, respectively), and between the content of bound lipids — inverse (r = –0.993;–0.995; –0.694).

STUDY OF LIPIDS, FATTY ACIDS AND LIPOPHILIC SUBSTANCES OF СONSOLIDA AMBIGUA (L.) P.W. BALL & HEYWOOD AND NIGELLA SATIVA L. SEEDS

The seeds of two medicinal plants from Ranunculaceae family – Consolida ambigua (L.) P.W. Ball & Heywood (Syn. Сonsolida ajacis Schur, ajacsova consolida, larkspur) and Nigella sativa L. (black cumin) cultivated in Uzbekistan was analyzed. Free and bound lipids were isolated from the seeds, the fatty acid composition of their neutral, glyco- and phospholipids was established. It was revealed that unsaturated components dominate among the ordinary fatty acids of seed lipids – oleic (C. ambigua) and linoleic (N. sativa). Their rare homologues – 11(Z)-eicosaenoic (C. ambigua) and 11,14(Z,Z)-eicosadienoic (Nigella sativa) acids were esterified mainly in the triacylglycerol molecules, and were found as free fatty acids of the studied oils. The major compounds among the 26 constituents of the essential oil of N. sativa seeds were p-cymene, terpinolene, β-pinene, limonene and sabinene.