A critical review of fear tests used on cattle, pigs, sheep, poultry and horses

n/

Anchoring mechanisms of membrane-associated M13 major coat protein

Bacteriophage M13 major coat protein is extensively used as a biophysical, biochemical, and molecular biology reference system for studying membrane proteins. The protein has several elements that control its position and orientation in a lipid bilayer. The N-terminus is dominated by the presence of negatively charged amino acid residues (Glu2, Asp4, and Asp5), which will always try to extend into the aqueous phase and therefore act as a hydrophilic anchor. The amphipathic and the hydrophobic transmembrane part contain the most important hydrophobic anchoring elements. In addition there are specific aromatic and charged amino acid residues in these domains (Phe 11, Tyr21, Tyr24, Trp26, Phe42, Phe45, Lys40, Lys43, and Lys44) that fine-tune the association of the protein to the lipid bilayer. The interfacial Tyr residues are important recognition elements for precise protein positioning, a function that cannot be performed optimally by residues with an aliphatic character. The Trp26 anchor is not very strong: depending on the context, the tryptophan residue may move in or out of the membrane. On the other hand, Lys residues and Phe residues at the C-terminus of the protein act in a unique concerted action to strongly anchor the protein in the lipid bilayer

Membrane assembly of M13 major coat protein: evidence for a structural adaptation in the hinge region and a titled transmembrane domain

New insights into the low-resolution Structure of the hinge region and the transmembrane domain of the membrane-bound major coat protein of the bacteriophage M13 are deduced from a single cysteine-scanning approach using fluorescence spectroscopy. New mutant coat proteins are labeled and reconstituted into phospholipid bilayers with varying headgroup compositions (PC, PE, and PG) and thicknesses (14:1PC, 18:1PC, and 22:1PC). Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. It is found that the protein is almost entirely embedded in the membrane, whereas the phospholipid headgroup composition of the membrane hardly affects the overall embedment of the protein in the membrane. From the assessment of a hydrophobic and hydrophilic face of the transmembrane helix, it is concluded that the helix is tilted with respect to the membrane normal. As compared to the thicker 18:1PC and 22:1PC membranes, reconstitution of the protein in the thin 14:1PC membranes results in a loss of helical structure and in the formation of a stretched conformation of the hinge region. It is suggested that the hinge region acts as a flexible spring between the N-terminal amphipathic arm and transmembrane hydrophobic helix. On average, the membrane-bound state of the coat protein can be seen as a gently curved and tilted, "banana-shaped" molecule, which is strongly anchored in the membrane-water interface at the C-terminus. From our experiments, we propose a rather small conformational adaptation of the major coat protein as the most likely reversible mechanism for responding to environmental changes during the bacteriophage disassembly and assembly process

Structural characterization of bacteriophage M13 solubilization by amphiphiles

The structural properties of bacteriophage M13 during disassembly were studied in different membrane model systems, composed of a homologue series of the detergents sodium octyl sulfate, sodium decyl sulfate, and sodium dodecyl sulfate. The structural changes during phage disruption were monitored by spin-labeled electron spin resonance (ESR) and circular dichroism spectroscopy. For the purpose of ESR spectroscopy the major coat protein mutants V31C and G38C were site-directed spin labeled in the intact phage particle. These mutants were selected because the mutated sites are located in the hydrophobic part of the protein, and provide good reporting locations for phage integrity. All amphiphiles studied were capable of phage disruption. However, no significant phage disruption was detected below the critical micelle concentration of the amphiphile used. Based on this finding and the linear dependence of phage disruption by amphiphiles on the phage concentration, it is suggested that the solubilization of the proteins of the phage coat by amphiphiles starts with an attachment to and penetration of amphiphile molecules into the phage particle. The amphiphile concentration in the phage increases in proportion to the amphiphile concentration in the aqueous phase. Incorporation of the amphiphile in the phage particle is accompanied with a change in local mobility of the spin-labeled part of the coat protein and its secondary structure. With increasing the amphiphile concentration in the phage particle, a concentration is reached where the concentration of the amphiphile in the aqueous phase is around its critical micelle concentration. A further increase in amphiphile concentration results in massive phage disruption. Phage disruption by amphiphiles appears to be dependent on the phage coat mutations. It is concluded that phage disruption is dependent on a hydrophobic effect, since phage solubilization could significantly be increased by keeping the hydrophilic part of the amphiphile constant, while increasing its hydrophobic part