Human apolipoprotein E N-terminal domain displacement of apolipophorin III from insect low density lipophorin creates a receptor-competent hybrid lipoprotein

The surface of Manduca sexta low density lipophorin (LDLp) particles was employed as a template to examine the relative lipid binding affinity of the 22 kDa receptor binding domain (residues 1–183) of human apolipoprotein E3 (apo E3). Isolated LDLp was incubated with exogenous apolipoprotein and, following re-isolation by density gradient ultracentrifugation, particle apolipoprotein content was determined. Incubation of recombinant human apo E3(1–183) with LDLp resulted in a saturable displacement of apolipophorin III (apo Lp-III) from the particle surface, creating a hybrid apo E3(1–183)-LDLp. Although subsequent incubation with excess exogenous apo Lp-III failed to reverse the process, human apolipoprotein A-I (apo A-I) effectively displaced apo E3(1–183) from the particle surface. We conclude that human apo E N-terminal domain possesses a higher intrinsic lipid binding affinity than apo Lp-III but has a lower affinity than human apo A-I. The apo E3(1–183)-LDLp hybrid was competent to bind to the low density lipoprotein receptor on cultured fibroblasts. The system described is useful for characterizing the relative lipid binding affinities of wild type and mutant exchangeable apolipoproteins and evaluation of their biological properties when associated with the surface of a spherical lipoprotein.     

Design,synthesis, and conformational studies of the hGM‐CSF derived peptide (13–27)–Gly–(75–87)

An analogue of the human granulocyte–macrophage colony‐stimulating factor (hGM‐CSF), hGM‐CSF(13–27)–Gly–(75–87) was synthesized by solid phase methodology. This analogue was designed to comprise helices A and C of the native growth factor, linked by a glycine bridge. Helices A and C form half of a four‐helix bundle motif in the crystal structure of the native factor and are involved in the interaction with α‐ and β‐chains of the heterodimeric receptor. A conformational analysis of the synthetic analogue by CD, two‐dimensional nmr spectroscopy, and molecular dynamics calculations is reported. The analogue is in a random structure in water and assumes a partially α‐helical conformation in a 1 : 1 trifluoroethanol/water mixture. The helix content in this medium is ∼ 70%. By 2D‐nmr spectroscopy, two helical segments were identified in the sequences corresponding to helices A and C. In addition to medium‐ and short‐range NOESY connectivities, a long‐range cross peak was found between the Cβ proton of Val¹⁶ and NH proton of His⁸⁷ (using the numbering of the native protein). Experimentally derived interproton distances were used as restraints in molecular dynamics calculations, utilizing the x‐ray coordinates as the initial structure. The final structure is characterized by two helical segments in close spatial proximity, connected by a loop region. This structure is similar to that of the corresponding domain in the x‐ray structure of the native growth factor in which helices A and C are oriented in an antiparallel fashion. The N‐terminal residues Gly–Pro of helix C are involved in an irregular turn connecting the two helical segments. As a consequence, helix C is appreciably shifted and slightly rotated with respect to helix A compared to the x‐ray structure of the native growth factor. These small differences in the topology of the two helices could explain the lower biological activity of this analogue with respect to that of the native growth factor. © 1999 John Wiley & Sons, Inc. Biopoly 50: 545–554, 1999     

High spatial resolution mass spectrometry imaging reveals the genetically programmed,developmental modification of the distribution of thylakoid membrane lipids among individual cells of maize leaf

Metabolism in plants is compartmentalized among different tissues, cells and subcellular organelles. Mass spectrometry imaging (MSI) with matrix‐assisted laser desorption ionization (MALDI) has recently advanced to allow for the visualization of metabolites at single‐cell resolution. Here we applied 5‐ and 10 μm high spatial resolution MALDI‐MSI to the asymmetric Kranz anatomy of Zea mays (maize) leaves to study the differential localization of two major anionic lipids in thylakoid membranes, sulfoquinovosyldiacylglycerols (SQDG) and phosphatidylglycerols (PG). The quantification and localization of SQDG and PG molecular species, among mesophyll (M) and bundle sheath (BS) cells, are compared across the leaf developmental gradient from four maize genotypes (the inbreds B73 and Mo17, and the reciprocal hybrids B73 × Mo17 and Mo17 × B73). SQDG species are uniformly distributed in both photosynthetic cell types, regardless of leaf development or genotype; however, PG shows photosynthetic cell‐specific differential localization depending on the genotype and the fatty acyl chain constituent. Overall, 16:1‐containing PGs primarily contribute to the thylakoid membranes of M cells, whereas BS chloroplasts are mostly composed of 16:0‐containing PGs. Furthermore, PG 32:0 shows genotype‐specific differences in cellular distribution, with preferential localization in BS cells for B73, but more uniform distribution between BS and M cells in Mo17. Maternal inheritance is exhibited within the hybrids, such that the localization of PG 32:0 in B73 × Mo17 is similar to the distribution in the B73 parental inbred, whereas that of Mo17 × B73 resembles the Mo17 parent. This study demonstrates the power of MALDI‐MSI to reveal unprecedented insights on metabolic outcomes in multicellular organisms at single‐cell resolution.     

Synthetic peptides mimicking the interleukin-6/gp130 interaction: a two-helix bundle system. Design and conformational studies.

The objective of our study was to mimic in a typical reductionist approach the molecular interactions observed at the interface between the gp130 receptor and interleukin-6 during formation of their complex. A peptide system obtained by reproducing some of the interleukin-6/gp130 molecular interactions into a two-helix bundle structure was investigated. The solution conformational features of this system were determined by CD and NMR techniques. The CD titration experiments demonstrated that the interaction between the designed peptides is specific and based on a well-defined stoichiometry. The NMR data confirmed some of the structural features of the binding mechanism as predicted by the rational design and indicated that under our conditions the recognition specificity and affinity can be explained by the formation of a two-helix bundle. Thus, the data reported herein represent a promising indication on how to develop new peptides able to interfere with formation of the interleukin-6/gp130 complex.     

Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize

Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII-LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII-LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.     

Comparative proteomic analysis of amaranth mesophyll and bundle sheath chloroplasts and their adaptation to salt stress

The effect of salt stress was analyzed in chloroplasts of Amaranthus cruentus var. Amaranteca, a plant NAD-malic enzyme (NAD-ME) type. Morphology of chloroplasts from bundle sheath (BSC) and mesophyll (MC) was observed by transmission electron microscopy (TEM). BSC and MC from control plants showed similar morphology, however under stress, changes in BSC were observed. The presence of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) was confirmed by immunohistochemical staining in both types of chloroplasts. Proteomic profiles of thylakoid protein complexes from BSC and MC, and their changes induced by salt stress were analyzed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (2-D BN/SDS-PAGE). Differentially accumulated protein spots were analyzed by LC–MS/MS. Although A. cruentus photosynthetic tissue showed the Kranz anatomy, the thylakoid proteins showed some differences at photosystem structure level. Our results suggest that A. cruentus var. Amaranteca could be better classified as a C₃–C₄ photosynthetic plant.     

Evaluation of the protein interfaces that form an intermolecular four-helix bundle as studied by computer simulation

Rational design of protein surface is important for creating higher order protein structures, but it is still challenging. In this study, we designed in silico the several binding interfaces on protein surfaces that allow a de novo protein–protein interaction to be formed. We used a computer simulation technique to find appropriate amino acid arrangements for the binding interface. The protein–protein interaction can be made by forming an intermolecular four-helix bundle structure, which is often found in naturally occurring protein subunit interfaces. As a model protein, we used a helical protein, YciF. Molecular dynamics simulation showed that a new protein–protein interaction is formed depending on the number of hydrophobic and charged amino acid residues present in the binding surfaces. However, too many hydrophobic amino acid residues present in the interface negatively affected on the binding. Finally, we found an appropriate arrangement of hydrophobic and charged amino acid residues that induces a protein–protein interaction through an intermolecular four-helix bundle formation.     

Ab initio prediction of the three-dimensional structure of a de novo designed protein: a double-blind case study

Ab initio structure prediction and de novo protein design are two problems at the forefront of research in the fields of structural biology and chemistry. The goal of ab initio structure prediction of proteins is to correctly characterize the 3D structure of a protein using only the amino acid sequence as input. De novo protein design involves the production of novel protein sequences that adopt a desired fold. In this work, the results of a double-blind study are presented in which a new ab initio method was successfully used to predict the 3D structure of a protein designed through an experimental approach using binary patterned combinatorial libraries of de novo sequences. The predicted structure, which was produced before the experimental structure was known and without consideration of the design goals, and the final NMR analysis both characterize this protein as a 4-helix bundle. The similarity of these structures is evidenced by both small RMSD values between the coordinates of the two structures and a detailed analysis of the helical packing.