Analysis of gated flux from or into sealed membrane fragments

Bernhardt J, Neumann E. Analysis of gated flux from or into sealed membrane fragments. Journal of Theoretical Biology. 1980;86(4):649-661.Physico-chemical factors that determine tracer substance flux from or into sealed vesicular structures are examined. Flux amplitudes are dependent on the average volume of a vesicle, while flux rates depend on the average number of transmembrane channels per vesicle. Gating processes leading to channel opening and/or closing affect both amplitudes and rates. Averaging over inhomogeneities in vesicle size and channel density leads to an explicit expression for time-dependent tracer content. Means for experimentally determining all variable factors in this expression are discussed

Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins

Amphipols (APols) are specially designed amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions in the absence of detergent. A8–35, a polyacrylate-based APol, has been grafted with an oligodeoxynucleotide (ODN). The synthesis, purification and properties of the resulting ‘OligAPol’ have been investigated. Grafting was performed by reacting an ODN carrying an amine-terminated arm with the carboxylates of A8–35. The use of OligAPol for trapping MPs and immobilizing them onto solid supports was tested using bacteriorhodopsin (BR) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) as model proteins. BR and OligAPol form water-soluble complexes in which BR remains in its native conformation. Hybridization of the ODN arm with a complementary ODN was not hindered by the assembly of OligAPol into particles, nor by its association with BR. BR/OligAPol and tOmpA/OligAPol complexes could be immobilized onto either magnetic beads or gold nanoparticles grafted with the complementary ODN, as shown by spectroscopic measurements, fluorescence microscopy and the binding of anti-BR and anti-tOmpA antibodies. OligAPols provide a novel, highly versatile approach to tagging MPs, without modifying them chemically nor genetically, for specific, reversible and targetable immobilization, e.g. for nanoscale applications

Pengaruh penambahan ekstrak daun beluntas dan azolla terhadap income over feed cost (iofc) dan nilai ekonomis pakan itik pedaging

Abstrak Tujuan dari penelitian ini adalah untuk mengetahui pengaruh penggunaan ekstrak daunbeluntas dan Azolla terhadap inccome over feed cost dan nilai ekonomis pakanitik pedaging. Materi yang digunakan dalam penelitian ini adalah 48 ekor itik pedaging dan bahan dengan komposisi sesuai dengan standar kebutuhan itik pedaging serta beluntas dan. Metode yang digunakan dalam penelitian ini adalah metode percobaan Lapang dengan menggunakan Rancangan Acak Lengkap (RAL) yaitu 4 perlakuan dan setiap perlakuan diulangi sebanyak 4 kali. Masing-masing percobaan diisi dengan 3 ekor itik. Perlakuan dalam penelitian ini adalah P0: (complete feed 100%), P1: complete feed 50% + ((azolla 85%) + (ekstrak bluntas 15%) 50%); P2: complete feed 50% +(( azolla 90%) + (ekstrak bluntas 10% )50%),  P3: complete feed 50% +(( azolla 95% )+ (ekstrak bluntas 5% )50%). Variabel yang diamati dalam penelitian ini adalah inccome over feed cost dan nilai ekonomis pakan itik pedaging, dan di analisis dengan menggunakan analisis ragam dengan rancangan Acak Lengkap (RAL). Apa bila antara perlakuan ada perbedaan, dilanjutkan dengan Uji BNT.Hasil penelitian menunjukan bahwa IOFC tertinggi pada perlakuan P0 dan  terendah pada perlakuan  P3. Dengan  nilai P0 sebesar Rp 6.262 dan nilai P3 sebesar Rp 605. Sedangkan Nilai ekonomis yang tertinggi terdapat pada kelompok P3  dan terendah pada P1 dengan  nilai P3 Rp 27.940 dan nilai P1 sebesar Rp 26.312.  Berdasarkan hasil penelitian disimpulkan bahwa pengguanaan ekstrak daun bluntas dan azolla sebagai pakan fermentasi sampai level 95% tidak memberikan pengaruh terhadap inccome over feed cost (IOFC), danNilai ekonomis pakan.     Abstract                     The  purpose of this research is to know the effect of using bluntas extract and Azolla as addition feed for inccome over feed cost and the economic value of ducks feed. The subject of this research were 48 ducks, complete feed, bluntas and azolla. Field exprerimental method was a method that was used in this research by using (completely randomized design) wherareas 4 treatments were applied. Each of treatment was repeated 4 times. The treatment of this researc were P0 : (complete feed 100%), P1: complete feed 50% +((azolla 85%)+(bluntas exstract 15%) 50%),  P2: complete feed 50%, +((azolla 90%)+(bluntas exstract 10%) 50%), P3: complete feed 50%, +((azolla 95%)+(bluntas exstract 5%) 50%). Variable of this study was income over feed cost and the economic value of ducks   feed. The result of this study showed that the higher income over feed founded on treatment of  P0 group with Rp. 2626 mean while the lowest on P2 group with Rp. 605. Wide result shows that the treatment give real effect (P<0,05) to IOFC of ducks. There was significant affect of IOFC and the economic value of boiler ducks feed. It was concluded that the combination of the level of protein interaction and Azolla and Bluntas. Obtained an IOFC of Rp 6.626, with production economic value of ducks feed of Rp 27. 940, at the age of  5 weeks Hbri ducks (P3

How Amphipols Embed Membrane Proteins: Global Solvent Accessibility and Interaction with a Flexible Protein Terminus

Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded β-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins

Statistical Mechanics of Membrane Protein Conformation: A Homopolymer Model

The conformation and the phase diagram of a membrane protein are investigated via grand canonical ensemble approach using a homopolymer model. We discuss the nature and pathway of $\alpha$-helix integration into the membrane that results depending upon membrane permeability and polymer adsorptivity. For a membrane with the permeability larger than a critical value, the integration becomes the second order transition that occurs at the same temperature as that of the adsorption transition. For a nonadsorbing membrane, the integration is of the first order due to the aggregation of $\alpha$-helices.Comment: RevTeX with 5 postscript figure

NMR Investigation of Structures of G-Protein Coupled Receptor Folding Intermediates

Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot et al., Biochemistry 29(1990), 4031) is postulated to proceed in 2 steps: Partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin et al., Biophys. J. 96(2009), 3187), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123 and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior

Statistical Mechanics of Integral Membrane Protein Assembly

During the synthesis of integral membrane proteins (IMPs), the hydrophobic amino acids of the polypeptide sequence are partitioned mostly into the membrane interior and hydrophilic amino acids mostly into the aqueous exterior. We analyze the minimum free energy state of polypeptide sequences partitioned into alpha-helical transmembrane (TM) segments and the role of thermal fluctuations using a many-body statistical mechanics model. Results suggest that IMP TM segment partitioning shares important features with general theories of protein folding. For random polypeptide sequences, the minimum free energy state at room temperature is characterized by fluctuations in the number of TM segments with very long relaxation times. Simple assembly scenarios do not produce a unique number of TM segments and jamming phenomena interfere with segment placement. For sequences corresponding to IMPs, the minimum free energy structure with the wildtype number of segments is free of number fluctuations due to an anomalous gap in the energy spectrum, and simple assembly scenarios produce this structure. There is a threshold number of random point mutations beyond which the size of this gap is reduced so that the wildtype groundstate is destabilized and number fluctuations reappear

Influence of the C‐terminus of the glycophorin A transmembrane fragment on the dimerization process

The monomer-dimer equilibrium of the glycophorin A (GpA) transmembrane (TM) fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix-helix packing in a membrane‐mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C‐terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement of TM fragments that takes place when leucine repeats are introduced in such protein segments. We also show that secondary structure of GpA derivatives was independent from dimerization, in agreement with the two‐stage model for membrane protein folding and oligomerization