Expression and purification of an engineered human endothelin receptor B in a monomeric form.

In humans, two endothelin receptors, ETa and ETb, are activated by three endogenous 21-mer cyclic peptides, ET-1, ET-2, and ET-3, which control various physiological processes, including vasoconstriction, vasodilation, and stimulation of cell proliferation. The first stage of this study it to produce a stable solubilized and purified receptor in a monodisperse state. This article is focused on the engineering, expression, purification, and characterization of the endothelin receptor B for subsequent structural and functional studies.

In meso structure of the cobalamin transporter, BtuB, at 1.95 A resolution.

Crystals of the apo form of the vitamin B12 and colicin receptor, BtuB, that diffract to 1.95 A have been grown by the membrane-based in meso technique. The structure of the protein differs in several details from that of its counterpart grown by the more traditional, detergent-based (in surfo) method. Some of these differences include (i) the five N-terminal residues are resolved in meso, (ii) residues 57-62 in the hatch domain and residues 574-581 in loop 21-22 are disordered in meso and are ordered in surfo, (iii) residues 278-287 in loop 7-8 are resolved in meso, (iv) residues 324-331 in loop 9-10, 396-411 in loop 13-14, 442-458 in loop 15-16 and 526-541 in loop 19-20 have large differences in position between the two crystal forms, as have residues 86-96 in the hatch domain, and (v) the conformation of residues 6 and 7 in the Ton box (considered critical to signal transduction and substrate transport) are entirely different in the two structures. Importantly, the in meso orientation of residues 6 and 7 is similar to that of the vitamin B12-charged state. These data suggest that the "substrate-induced" 180 degrees -rotation of residues 6 and 7 reported in the literature may not be a unique signalling event. The extent to which these findings agree with structural, dynamic and functional insights gleaned from site-directed spin labelling and electron paramagnetic resonance measurements is evaluated. Packing in in meso grown crystals is dense and layered, consistent with the current model for crystallogenesis of membrane proteins in lipidic mesophases. Layered packing has been used to locate the transmembrane hydrophobic surface of the protein. Generally, this is consistent with tryptophan, tyrosine, lipid and CalphaB-factor distributions in the protein, and with predictions based on transfer free energy calculations.

Transmembrane peptides stabilize inverted cubic phases in a biphasic length-dependent manner: implications for protein-induced membrane fusion.

WALP peptides consist of repeating alanine-leucine sequences of different lengths, flanked with tryptophan "anchors" at each end. They form membrane-spanning alpha-helices in lipid membranes, and mimic protein transmembrane domains. WALP peptides of increasing length, from 19 to 31 amino acids, were incorporated into N-monomethylated dioleoylphosphatidylethanolamine (DOPE-Me) at concentrations up to 0.5 mol % peptide. When pure DOPE-Me is heated slowly, the lamellar liquid crystalline (L(alpha)) phase first forms an inverted cubic (Q(II)) phase, and the inverted hexagonal (H(II)) phase at higher temperatures. Using time-resolved x-ray diffraction and slow temperature scans (1.5 degrees C/h), WALP peptides were shown to decrease the temperatures of Q(II) and H(II) phase formation (T(Q) and T(H), respectively) as a function of peptide concentration. The shortest and longest peptides reduced T(Q) the most, whereas intermediate lengths had weaker effects. These findings are relevant to membrane fusion because the first step in the L(alpha)/Q(II) phase transition is believed to be the formation of fusion pores between pure lipid membranes. These results imply that physiologically relevant concentrations of these peptides could increase the susceptibility of biomembrane lipids to fusion through an effect on lipid phase behavior, and may explain one role of the membrane-spanning domains in the proteins that mediate membrane fusion.

The Membrane Protein Data Bank.

The Membrane Protein Data Bank (MPDB) is an online, searchable, relational database of structural and functional information on integral, anchored and peripheral membrane proteins and peptides. Data originates from the Protein Data Bank and other databases, and from the literature. Structures are based on X-ray and electron diffraction, nuclear magnetic resonance and cryoelectron microscopy. The MPDB is searchable online by protein characteristic, structure determination method, crystallization technique, detergent, temperature, pH, author, etc. Record entries are hyperlinked to the PDB and Pfam for viewing sequence, three-dimensional structure and domain architecture, and for downloading coordinates. Links to PubMed are also provided. The MPDB is updated weekly in parallel with the Protein Data Bank. Statistical analysis of MPDB records can be performed and viewed online. A summary of the statistics as applied to entries in the MPDB is presented. The data suggest conditions appropriate for crystallization trials with novel membrane proteins.

Strength of thermal undulations of phospholipid membranes.

The temperature dependence of intermembrane interactions in freely suspended multilamellar membranes of dimiristoylphosphatidylcholine in D2O was studied using small-angle neutron scattering (SANS) and high-resolution x-ray diffraction (HRXRD). The study reveals that the Helfrich's undulation force is the dominating repulsion force at temperatures above 48.6 degrees C and intermembrane distances larger than 20.5 A. At approximately 77 degrees C the onset of the unbinding transition in the multilamellar membranes is observed. This transition has a continuous behavior in agreement with theoretical predictions and proceeds in accordance with a two-state model. Complimentary analysis of SANS and HRXRD data permits accurate calculation of the fundamental undulation force constant cfl. The obtained value of cfl=0.111+/-0.005 is in good agreement with theoretical calculations. The results of this work demonstrate a key role of Helfrich's undulations in the balance of intermembrane interactions of lipid membranes under physiological temperatures and suggest that thermal undulations play an important part in the interactions of biological membranes. The agreement of the predictions with the experimental data confirms that lipid membranes can be considered as random fluctuating surfaces that can be described well by current theoretical models and that they can serve as a powerful tool for studying behavior of such surfaces.

The kinetics of non-lamellar phase formation in DOPE-Me: relevance to biomembrane fusion.

The mechanism of the lamellar/inverted cubic (QII) phase transition is related to that of membrane fusion in lipid systems. N-Monomethylated dioleoylphosphatidylethanolamine (DOPE-Me) exhibits this transition and is commonly used to investigate the effects of exogenous substances, such as viral fusion peptides, on the mechanism of membrane fusion. We studied DOPE-Me phase behavior as a first step in evaluating the effects of membrane-spanning peptides on inverted phase formation and membrane fusion. These measurements show that: a) the onset temperatures for QII and inverted hexagonal (HII) phase formation both are temperature scan rate-dependent; b) longer pre-incubation times at low temperature and lower temperature scan rates favor formation of the QII phase; and c) in temperature-jump experiments between 61 and 65 degrees C, the meta-stable HII phase forms initially, and disappears slowly while the QII phase develops. These observations are rationalized in the context of a mechanism for both the lamellar/non-lamellar phase transition and the related process of membrane fusion.

Crystallization screens: compatibility with the lipidic cubic phase for in meso crystallization of membrane proteins.

The in meso method for growing crystals of membrane proteins uses a spontaneously forming lipidic cubic mesophase. The detergent-solubilized protein is dispersed with lipid, typically monoolein, and in so doing the cubic phase self-assembles. A precipitant is added to trigger crystal nucleation and growth. The commercial screen solution series are convenient for use in crystallization trials. The aim of this study was to determine which of the Hampton Screen and Screen 2 series of solutions are compatible with the in meso method. These screens contain components any of which could destroy the cubic phase. X-ray diffraction was used for phase identification and for microstructure characterization. The study was done at 4 degrees C and at 20 degrees C. Two types of sample preparations were examined. One used an excess of half-strength screen solution (Prep. 1). The other used a limiting quantity of undiluted screen solution (Prep. 2). At 20 degrees C, over 90% of the screen solutions produced the cubic phase with Prep. 1. This figure dropped to 50% with Prep. 2. In contrast, 50 to 60% of the screens were cubic phase compatible at 4 degrees C under Prep. 1 conditions. The figure fell to 25% with Prep. 2. The mode of action of the diverse screen components are explained on the basis of the phase properties of the monoolein/water system.

Biophysics and synchrotron radiation where the marriage fails. X-ray damage of lipid membranes and mesophases.

The call for brighter synchrotron X-radiation sources for use in structural biology research is barely audible as we enter the new millennium. Our brightest sources are already creating havoc when used at design specifications because of radiation damage. The time is long overdue to take stock of where we are and where we wish to go with regards to using existing sources and to designing new ones. The problem of radiation damage is particularly severe in studies involving kinetics and mechanism where cryotechniques are not always viable. Accordingly, we need to understand the very nature of radiation damage and to devise means for minimizing it. This is the thrust of the current study as applied to lipid membranes and mesophases. Here, we report on two very different types of radiation damage. One involves a dramatic phase transformation and the other a disordering of lamellar stacking. How beam energy and dose/rate affect damage is also discussed. The work highlights the nature of the damage process and the need for additional studies if we are to make most efficient use of an important resource, synchrotron radiation.

Investigation of the structure of thylakoid membranes (spinach) by means of small-angle neutron scattering.

This paper presents experimental data on the determination of the thickness of thylakoid membranes by small-angle neutron scattering. The thylakoids were isolated from spinach chloroplasts. The partial volume of proteins and lipids in the "washed" and "unwashed" membranes was estimated. It is shown that the thickness of thylakoid membranes, measured with this techniques depends on the way the membranes were separated. When isolated thylakoids by the standard method, the membrane thickness amounted to 75 A but if the extracted thylakoids were additionally washed with the isolation medium, the measured thickness was 50 A. In this case a significant decrease in the protein partial volume of the membrane was observed. The obtained results make it possible to explain numerous data on X-ray and small-angle neutron scattering by thylakoid membranes of different origins, proceeding from the assumption that all these membranes have a unified structure and consist of a stable core with a thickness of about 50 A, and layers of peripheral, weakly bound proteins with a thickness which may depends on the nature of the object under investigation and extracting conditions.

Mathematical model of the cut-off effect in the homologous series of tertiary amine local anesthetics.

It is proposed a simple mathematical model explaining the quasi parabolic dependence of the local anesthetic activity on the length of hydrophobic substituent in the homologous series of tertiary amines (TA). It is suggested that the molecules of TA intercalate between the lipid molecules in bilayers. Due to the mismatch between the lengths of lipid and TA hydrocarbon chains the intercalation results in a decrease in the bilayer thickness. The quasi parabolic dependence is the result of combination of partition equilibria and of geometrical parameters of interacting molecules in the bilayer. The model predicts that the TA chain length at which the maximum activity is observed should be dependent on the lipid: aqueous phase volume ratio. The empirical parameters used in the model are obtained from the X-ray diffraction on multilamellar egg yolk phosphatidylcholine (EYPC) dispersions with the monohydrochloride of [2-(heptyloxy)phenyl]-2-(1-piperidinyl)ethyl ester of carbamic acid and from the partition equilibria of its alkyloxy homologs with unilamellar EYPC liposomes.