Biophysical characterization and stabilization of detergent-solubilized lipoprotein N-acyl transferase from P. aeruginosa and E. coli.

Lipoproteins are important for bacterial growth and virulence and interest in them as targets for antibiotic development is growing. Lipoprotein N-acyl transferase (Lnt) catalyzes the final step in the lipoprotein posttranslational processing pathway. The mature lipoprotein can remain in the inner membrane or be trafficked to the outer membrane in the case of diderm prokaryotes. With a view to obtaining high-resolution crystal structures of membrane integral Lnt for use in drug discovery a program was undertaken to generate milligram quantities of stable, homogenous and functional protein. This involved screening across bacterial species for suitable orthologues and optimization at the level of protein expression, solubilization and stability. Combining biophysical and functional characterization, orthologous Lnt from Escherichia coli and the opportunistic human pathogen Pseudomonas aeruginosa was identified as suitable for the proposed structure determination campaign that ultimately yielded crystal structures. The rational approaches taken that eventually provided structure-quality protein are presented in this report.

High resolution 1H NMR of a lipid cubic phase using a solution NMR probe.

The cubic mesophase formed by monoacylglycerols and water is an important medium for the in meso crystallogenesis of membrane proteins. To investigate molecular level lipid and additive interactions within the cubic phase, a method was developed for improving the resolution of (1)H NMR spectra when using a conventional solution state NMR probe. Using this approach we obtained well-resolved J-coupling multiplets in the one-dimensional NMR spectrum of the cubic-Ia3d phase prepared with hydrated monoolein. A high resolution t-ROESY two-dimensional (1)H NMR spectrum of the cubic-Ia3d phase is also reported. Using this new methodology, we have investigated the interaction of two additive molecules, L-tryptophan and ruthenium-tris(2,2-bipyridyl) dichloride (rubipy), with the cubic mesophase. Based on the measured chemical shift differences when changing from an aqueous solution to the cubic phase, we conclude that L-tryptophan experiences specific interactions with the bilayer interface, whereas rubipy remains in the aqueous channels and does not associate with the lipid bilayer.

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.

Controlling release from the lipidic cubic phase. Amino acids, peptides, proteins and nucleic acids.

Drugs are optimally effective in the therapeutic concentration range. A challenge in the delivery area is to design a system that will allow the therapeutic range to be accessed and to be maintained for defined periods. The lipidic cubic phases have been used as delivery matrices with such properties. For water-soluble drugs, release from the cubic phase is controlled by transport through aqueous channels that permeate the phase. Channel size can be tuned over wide limits by adjusting temperature and lipid identity. Thus, the possibility exists to regulate the rate of drug release from the cubic phase. With a view to exploiting these features for small molecule, proteinaceous and nucleic acid drugs, we have taken a systematic approach toward understanding how cubic phase transport is controlled by phase identity and microstructure and by the physical and chemical properties of the drug itself. Measurements were made using tryptophan, rubipy, DNA and six proteins as drug surrogates and with three hosting lipids. Remarkably, transport was observed with apo-ferritin whose size far exceeds that of the aqueous channel suggesting a molecular breathing or peristalsis type of facilitated release. Exquisite control over release was achieved by adjusting electrostatic interaction strength and by His-tag displacement.

Gramicidin structure and disposition in highly curved membranes.

With a view to deciphering aspects of the mechanism of membrane protein crystallization in lipidic mesophases (in meso crystallization), an examination of the structure and disposition of the pore-forming peptide, gramicidin, in the lipidic cubic phase was undertaken. At its simplest, the cubic phase consists of lipid and water in the form of a molecular 'sponge.' The lipid exists as a continuous, highly curved bilayer that divides the aqueous component into two interpenetrating but non-contacting channels. In this study, we show that gramicidin reconstitutes into the lipid bilayer of the cubic phase and that it adopts the channel, or helical dimer, conformation therein. Fluorescence quenching with brominated lipid was used to establish the bilayer location of the peptide. Electronic absorption and emission spectroscopies corroborated this finding. Peptide conformation in the cubic phase membrane was determined by circular dichroism. The identity and microstructure of the mesophases, and their capacity to accommodate gramicidin and other system components (sodium dodecyl sulfate, trifluoroethanol), was established by small-angle X-ray diffraction. Beyond a limiting concentration, gramicidin destabilized the cubic phase in favor of the inverted hexagonal phase. While gramicidin remained bilayer bound as membrane thickness changed, its conformation responded to the degree of bilayer mismatch with the hydrophobic surface of the peptide. These findings support the hypothesis that reconstitution into the lipid bilayer is an integral part of the in meso crystallization process as applied to membrane proteins. They also suggest ways for improving the process of membrane protein crystallogenesis.

Controlling release from the lipidic cubic phase by selective alkylation.

The lipidic cubic phase can be viewed as a molecular sponge consisting of interpenetrating nanochannels filled with water and coated by lipid bilayers. It has been used as a delivery matrix for low-molecular-weight drugs. For those that are water-soluble, release is fast and unregulated. This study seeks to exploit the lipid bilayer compartment as a location within the cubic phase in which to 'hydrophobically' anchor the water-soluble drug. This was accomplished by controlling partitioning into, and thus release from, the aqueous compartment of the cubic phase. Tryptophan was used as a surrogate water-soluble drug and alkylation was implemented to regulate release. By adjusting alkyl chain length, exquisite control was realized. Without alkylation, 20% of the tryptophan was released under standard conditions (infinite sink with a 30-mg cubic phase source at pH 7 and 20 degrees C) over a period of 30 min (t(20)). In the case of derivatives with alkyl chains two and eight carbon atoms long, t(20) values of 3 and 13 days, respectively, were observed. Eliminating the charge on tryptophan completely by alkylation produced a derivative that became irreversibly lodged in the lipid bilayer. The release behavior of the short-chain derivatives was mathematically modeled and parameters describing transport have been obtained. Cubic phase partition coefficients for tryptophan and its derivatives were measured to facilitate modeling. The implications of these findings with regard to the cubic phase and related delivery systems, and to vaccine efficacy are discussed.

Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization.

The in meso method for membrane protein crystallization uses a lipidic cubic phase as the hosting medium. The cubic phase provides a lipid bilayer into which the protein presumably reconstitutes and from which protein crystals nucleate and grow. The solutions used to spontaneously form the protein-enriched cubic phase often contain significant amounts of detergents that were employed initially to purify and to solubilize the membrane protein. By virtue of their surface activity, detergents have the potential to impact on the phase properties of the in meso system and, by extension, the outcome of the crystallization process. The purpose of this study was to quantify the effects that a popular series of nonionic detergents, the n-alkyl-beta-D-glucopyranosides, have on the phase behavior of hydrated monoolein, the lipid upon which the in meso method is based. Phase identity and phase microstructure were characterized by small-angle x-ray diffraction on samples prepared to mimic in meso crystallization conditions. Measurements were made in the 0-40 degrees C range. Samples prepared in the cooling direction allow for the expression of metastability, a feature of liquid crystalline phases that might be exploited in low-temperature crystallization. The results show that the cubic phase is relatively insensitive to small amounts of alkyl glucosides. However, at higher levels the detergents trigger a transition to the lamellar phase in a temperature- and salt concentration-dependent manner. These effects have important implications for in meso crystallization. A diffraction-based method for assaying detergents is presented.

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.

Molecular mechanism for the crystallization of bacteriorhodopsin in lipidic cubic phases.

Crystals of transmembrane proteins may be grown from detergent solutions or in a matrix of membranous lipid bilayers existing in a liquid crystalline state and forming a cubic phase (in cubo). While crystallization in micellar solutions appears analogous to that for soluble proteins, crystallization in lipidic matrices is poorly understood. As this method was shown to be applicable to several membrane proteins, understanding its mechanism will facilitate a rational design of crystallization, minimizing the laborious screening of a large number of parameters. Using polarization microscopy and low-angle X-ray diffraction, experimental evidence is provided to support a mechanistic model for the in cubo crystallization of bacteriorhodopsin in a lipid matrix. Membrane proteins are thought to reside in curved lipid bilayers, to diffuse into patches of lower curvature and to incorporate into lattices which associate to form highly ordered three-dimensional crystals. Critical testing of this model is necessary to generalize it to other membrane proteins.

Heparin binding by the HIV-1 tat protein transduction domain.

The protein transduction domain from the HIV-1 tat protein (termed PTD-tat) has been fused to the C-terminus of a model cargo protein, the IgG binding domain of streptococcal protein G. We demonstrate that PG-Ctat (PTD-tat fused to the C-terminus of protein G) binds to a heparin affinity column. PG-Ctat binds with relatively high affinity, as shown by its elution at 1.6 M NaCl. The heparin binding properties of PTD-tat are consistent with the idea that heparan sulfate, an analog of heparin found at the cell surface, plays a role in the translocation of PTD-tat fusions. We suggest that the heparin-binding properties of PTD-tat can be exploited for purification of PTD-tat fusions in the absence of affinity tags.

Magnetic susceptibility tensor and heme contact shifts determinations in the Rhodobacter capsulatus ferricytochrome c': NMR and magnetic susceptibility studies.

The 1H and 15N resonances of the carbon monoxide complex of ferrocytochrome c' of Rhodobacter capsulatus, a ferrous diamagnetic heme protein, have been extensively assigned by TOCSY-HSQC, NOESY-HSQC, and HSQC-NOESY-HSQC 3D heteronuclear experiments performed on a 7 mM sample labeled with 15N. Based on short-range and medium-range NOEs and H(N) exchange rates, the secondary structure consists of four helices: helix 1 (3-29), helix 2 (33-48), helix 3 (78-101), and helix 4 (103-125). The 15N, 1HN, and 1H(alpha) chemical shifts of the CO complex form are compared to those of the previously assigned oxidized (or ferric) state. From the chemical shift differences between these redox states, the orientation and the anisotropy of the paramagnetic susceptibility tensor have been determined using the crystallographic coordinates of the ferric state. The chi-tensor is axial, and the orientation of the z-axis is approximately perpendicular to the heme plane. The paramagnetic chemical shifts of the protons of the heme ligand have been determined and decomposed into the Fermi shift and dipolar shift contributions. Magnetic susceptibility studies in frozen solutions have been performed. Fits of the susceptibility data using the model of Maltempo (Maltempo, M. M. J. Chem. Phys. 1974, 61, 2540-2547) are consistent with a rather low contribution of the S = 3/2 spin state over the range of temperatures and confirm the value of the axial anisotropy. Values in the range 10.4-12.5 cm(-1) have been inferred for the axial zero-field splitting parameter (D). Analysis of the contact shift and the susceptibility data suggests that cytochrome c' of Rb. capsulatus exhibits a predominant high-spin character of the iron in the oxidized state at room temperature.

Rational design of lipid molecular structure: a case study involving the C19:1c10 monoacylglycerol.

The phase properties of lipids have far-reaching consequences in membrane biology. Their influence ranges from domain formation in intact biomembranes to membrane protein reconstitution and crystallization. To exploit phase behavior in the spirit of rational design, it is imperative that the rules relating lipid molecular structure and liquid crystal or mesophase behavior be established. Phase behavior is quantitatively and concisely represented in the form of temperature-composition phase diagrams. A somewhat limited number of phase diagrams exists for the monoacylglycerols. The objective of the current study was to determine the quality of phase behavior prediction for a specific monoacylglycerol based on an analysis of the existing phase diagrams for related chain homologs. To this end, a phase diagram for the monononadecenoin (19:1c10)/water system was predicted in the temperature range from -15 degrees C to 120 degrees C and from 0% to 80% (w/w) water. The prediction was tested by constructing the corresponding phase diagram using low- and wide-angle x-ray diffraction, differential scanning calorimetry, and polarized light microscopy. The results show that the predicted and experimental phase diagrams agree remarkably well. They also highlight the need for additional phase studies of the type described to enlarge the data bank of phase diagrams and to strengthen the foundations of the rational design approach.

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.

Model for the structure of the HIV gp41 ectodomain: insight into the intermolecular interactions of the gp41 loop.

In human immunodeficiency virus (HIV) the viral envelope proteins gp41 and gp120 form a non-covalent complex, which is a potential target for AIDS therapies. In addition gp41 plays a possible role in HIV infection of B cells via the complement system. In an effort to better understand the molecular interactions of gp41, the structure of the HIV gp41 ectodomain has been modeled using the NMR restraints of the simian immunodeficiency virus (SIV) gp41 ectodomain (M. Caffrey, M. Cai, J. Kaufman, S.J. Stahl, P.T. Wingfield, A.M. Gronenborn, G.M. Clore, Solution structure of the 44 kDa ectodomain of SIV gp41, EMBO J. 17 (1998) 4572--4584). The resulting model presents the first structural information for the HIV gp41 loop, which has been implicated to play a direct role in binding to gp120 and C1q of the complement system.

Phase behavior of a monoacylglycerol: (myverol 18-99K)/water system.

The phase behavior of Myverol 18-99K, a food emulsifier rich in monoacylglycerols, in combination with water has been determined. X-ray diffraction and polarized light microscopy (PLM) were used for phase identification and structure characterization. Phase behavior was established in the temperature range from -15 to 50 degrees C and in the composition range from dry to full hydration. Phases identified include the solid lamellar crystal (Lc) phase, the liquid fluid isotropic phase and three liquid crystal phases, the lamellar liquid crystal, the cubic-Ia3d and the cubic-Pn3m phase. Phase information is reported in the form of temperature-composition phase diagrams. It was collected under equilibrium conditions where measurements were made in the heating direction beginning with the Lc phase at -15 degrees C. Phase metastability was also examined in which the natural tendency of the liquid crystal phases to undercool was facilitated. Under this condition, both cubic phases were found to remain free of the solid Lc phase over a relatively wide range of hydration values down to 0 degrees C. The microstructure of the different phases and its dependence on temperature and hydration has been determined. Compositional analysis using thin layer chromatography and gas chromatography/mass spectrometry shows that Myverol 18-99K consists of 82% monoacylglycerols (86.6% monoolein, 7. 0% monostearin, 3.5% monopalmitin, 0.9% monoarachidin, 2.0% unidentified). The equilibrium and metastable phase diagrams of the Myverol 18-99K/water system show remarkable similarity to those reported for the monoolein/water system (Qiu, H., Caffrey, M., 2000. The phase diagram of the monoolein/water system: metastability and equilibrium aspects Biomaterials 21, 223-594.).

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.

A lipid's eye view of membrane protein crystallization in mesophases.

Lipidic mesophases have been used to produce diffraction-quality crystals of several membrane proteins. The mechanism by which the method works is a mystery. The thrust is to continue to use it whilst deciphering the underlying mechanism and solving the second 'phase problem' in membrane protein crystallography. The method, which probably shares similarities with crystal growth in microgravity, is examined here from a lipid and a phase science perspective.

Membrane protein crystallization in lipidic mesophases: detergent effects.

The "cubic phase method" for growing crystals of membrane proteins uses a complex mixture of water, lipid, protein, and other components. The current view is that the cubic phase is integral to the process. Thus additives from whatever source introduce the possibility of destabilizing the phase, thereby compromising the crystallization process. Detergents are used to solubilize membrane proteins and are likely to be ported into the cubic medium with the target protein. Depending on the identity and concentration of the detergent, the cubic phase, which itself is membranous, may be solubilized or destabilized in such a way as to render it unsuitable as a crystal growing system. The nonionic detergent n-dodecyl-beta-D-maltopyranoside is commonly used in membrane protein work. In this study, we evaluate its effect on the cubic mesophase of hydrated monoolein. X-ray diffraction was used for phase identification and mesophase microstructure characterization. The results show that while low levels of the detergent are tolerated, increasing concentrations trigger a cubic-to-lamellar phase transition in a temperature-dependent manner. This finding is rationalized in the context of complementary molecular shapes of the lipid and the detergent and has implications for the mechanism of crystallization in lipidic mesophases as discussed.