Pharmacophore-based screening and modification of amiloride analogs for targeting the NhaP-type cation-proton antiporter in Vibrio cholerae.

The genome of Vibrio cholerae contains three structural genes for the NhaP-type cation-proton antiporter paralogues, Vc-NhaP1, Vc-NhaP2, and Vc-NhaP3, mediating exchange of K+ and or Na+ for protons across the membrane. Based on phenotypic analysis of chromosomal Vc-NhaP1, Vc-NhaP2, and Vc-NhaP3 triple deletion mutants, we suggest that Vc-NhaP paralogues are primarily K+/H+ antiporters and might play a role in the acid tolerance response of V. cholerae as it passes through the gastric acid barrier of the stomach. Comparison of the biochemical properties of Vc-NhaP isoforms revealed that Vc-NhaP2 was the most active among all three paralogues. Therefore, the Vc-NhaP2 antiporter is a plausible therapeutic target for developing novel inhibitors targeting these ion exchangers. Our structural and mutational analysis of Vc-NhaP2 identified a putative cation-binding pocket formed by antiparallel extended regions of two transmembrane segments (TMSs V and XII) along with TMS VI. Molecular dynamics simulations suggested that the flexibility of TMSs V and XII is crucial for intramolecular conformational events in Vc-NhaP2. In this study, we developed putative Vc-NhaP2 inhibitors from amiloride analogs. Molecular docking of the modified amiloride analogs revealed promising binding properties. The four selected drugs potentially interacted with functionally important amino acid residues located on the cytoplasmic side of TMS VI, the extended chain region of TMSs V and XII, and the loop region between TMSs VIIII and IX. Molecular dynamics simulations revealed that binding of the selected drugs can potentially destabilize Vc-NhaP2 and alter the flexibility of functionally important TMS VI. This work presents the utility of in silico approaches for the rational identification of potential targets and drugs that could target NhaP2 cation proton antiporters to control V. cholerae. The goal was to identify potential drugs that could be validated in future experiments.

Solution structure of the cytoplasmic domain of NhaP2 a K+/H+ antiporter from Vibrio cholera.

NhaP2 is a K+/H+ antiporter from Vibrio cholerae which consists of a transmembrane domain and a cytoplasmic domain of approximately 200 amino acids, both of which are required for cholera infectivity. Here we present the solution structure for a 165 amino acid minimal cytoplasmic domain (P2MIN) form of the protein. The structure reveals a compact N-terminal domain which resembles a Regulator of Conductance of K+ channels (RCK) domain connected to a more open C-terminal domain via a flexible 20 amino acid linker. NMR titration experiments showed that the protein binds ATP through its N-terminal domain, which was further supported by waterLOGSY and Saturation Transfer Difference NMR experiments. The two-domain organisation of the protein was confirmed by BIOSAXS, which also revealed that there are no detectable-ATP-induced conformational changes in the protein structure. Finally, in contrast to all known RCK domain structures solved to date, the current work shows that the protein is a monomer.

Solution structure and oligomeric state of the E. coliglycerol facilitator.

Protein dynamics at atomic resolution can provide deep insights into the biological activities of proteins and enzymes but they can also make structure and dynamics studies challenging. Despite their well-known biological and pharmaceutical importance, integral membrane protein structure and dynamics studies lag behind those of water-soluble proteins mainly owing to solubility problems that result upon their removal from the membrane. Escherichia coli glycerol facilitator (GF) is a member of the aquaglyceroporin family that allows for the highly selective passive diffusion of its substrate glycerol across the inner membrane of the bacterium. Previous molecular dynamics simulations and hydrogen-deuterium exchange studies suggested that protein dynamics play an important role in the passage of glycerol through the protein pore. With the aim of studying GF dynamics by solution and solid-state nuclear magnetic resonance (NMR) spectroscopy we optimized the expression of isotope-labelled GF and explored various solubilizing agents including detergents, osmolytes, amphipols, random heteropolymers, lipid nanodiscs, bicelles and other buffer additives to optimize the solubility and polydispersity of the protein. The GF protein is most stable and soluble in lauryl maltose neopentyl glycol (LMNG), where it exists in a tetramer-octamer equilibrium. The solution structures of the GF tetramer and octamer were determined by negative-stain transmission electron microscopy (TEM), size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) and solid-state magic-angle spinning NMR spectroscopy. Although NMR sample preparation still needs optimization for full structure and dynamics studies, negative stain TEM and SEC-SAXS revealed low-resolution structures of the detergent-solubilized tetramer and octamer particles. The non-native octamer appears to form from the association of the cytoplasmic faces of two tetramers, the interaction apparently mediated by their disordered N- and C-termini. This information may be useful in future studies directed at reducing the heterogeneity and self-association of the protein.

Physiological, Structural, and Functional Analysis of the Paralogous Cation-Proton Antiporters of NhaP Type from Vibrio cholerae.

The transmembrane K+/H+ antiporters of NhaP type of Vibrio cholerae (Vc-NhaP1, 2, and 3) are critical for maintenance of K+ homeostasis in the cytoplasm. The entire functional NhaP group is indispensable for the survival of V. cholerae at low pHs suggesting their possible role in the acid tolerance response (ATR) of V. cholerae. Our findings suggest that the Vc-NhaP123 group, and especially its major component, Vc-NhaP2, might be a promising target for the development of novel antimicrobials by narrowly targeting V. cholerae and other NhaP-expressing pathogens. On the basis of Vc-NhaP2 in silico structure modeling, Molecular Dynamics Simulations, and extensive mutagenesis studies, we suggest that the ion-motive module of Vc-NhaP2 is comprised of two functional regions: (i) a putative cation-binding pocket that is formed by antiparallel unfolded regions of two transmembrane segments (TMSs V/XII) crossing each other in the middle of the membrane, known as the NhaA fold; and (ii) a cluster of amino acids determining the ion selectivity.

Altered rPrP substrate structures and their influence on real-time quaking induced conversion reactions.

BACKGROUND: Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrPd). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay. No harmonized method for producing the protein has been agreed upon, in part due to the variety of substrates that have been applied in RT-QuIC. METHODS: This study examines the effects of two different rPrP refolding protocols on the production, QuIC performance, and structure characteristics of two constructs of rPrP commonly used in QuIC: full length hamster and a sheep-hamster chimeric rPrP. RESULTS: Under the described conditions, the best performing substrate was the chimeric sheep-hamster rPrP produced by shorter guanidine-HCl exposure and faster gradient elution. CONCLUSIONS: The observation that different rPrP production protocols influence QuIC performance indicates that caution should be exercised when comparing inter-laboratory QuIC results.

Nanoscale Assembly of High-Mobility Group AT-Hook 2 Protein with DNA Replication Fork.

High mobility group AT-hook 2 (HMGA2) protein is composed of three AT-hook domains. HMGA2 expresses at high levels in both embryonic stem cells and cancer cells, where it interacts with and stabilizes replication forks (RFs), resulting in elevated cell proliferation rates. In this study, we demonstrated that HMGA2 knockdown reduces cell proliferation. To understand the features required for interaction between HMGA2 and RFs, we studied the solution structure of HMGA2, free and in complex with RFs, using an integrated host of biophysical techniques. Circular dichroism and NMR experiments confirmed the disordered state of unbound HMGA2. Dynamic light scattering and sedimentation velocity experiments demonstrated that HMGA2 and RF are monodisperse in solution, and form an equimolar complex. Small-angle x-ray scattering studies revealed that HMGA2 binds in a side-by-side orientation to RF where 3 AT-hooks act as a clamp to wrap around a distorted RF. Thus, our data provide insights into how HMGA2 interacts with stalled RFs and the function of the process.

The Dynamic Landscape of the Full-Length HIV-1 Transactivator of Transcription.

The type 1 human immunodeficiency virus (HIV-1) transactivator of transcription (Tat) is a small RNA-binding protein essential for viral gene expression and replication. It has also been shown to bind to a large number of human proteins and to modulate many different cellular activities. We have used nuclear magnetic resonance (NMR) spectroscopy and hydrogen exchange chemistry to measure backbone dynamics over the millisecond to picosecond time scales. Sequential backbone assignment was facilitated by several isotope labeling schemes, including uniform labeling, site-specific labeling, and unlabeling. (15)N NMR relaxation parameters were measured and analyzed by reduced spectral density mapping and the Lipari-Szabo Model-Free approach to characterize the backbone dynamics on the picosecond to nanosecond time scale. The results indicate that the protein exists in an extended disordered conformational ensemble. NMR relaxation dispersion profiles show that on the millisecond time scale no conformational exchange is detected for any of the residues, supporting the model of a disordered backbone. NMR chemical shift differences from random coil values suggest that some segments of the protein have a modest propensity to fold; comparison to X-ray diffraction structures of Tat complexes indicates that some segments of the protein function through an induced-fit mechanism whereas other segments likely operate by conformational selection. Surprisingly, measured hydrogen exchange rates are higher than predicted for a disordered polymer, but this is explained as being caused by the high net charge on the protein that enhances base-catalyzed hydrogen exchange. The dynamics results provide a deeper understanding of the protein conformational ensemble and form a foundation for future studies of the conformational changes that accompany the formation of the superelongation complex that activates viral transcription.

Folding and stability of the aquaglyceroporin GlpF: Implications for human aqua(glycero)porin diseases.

Aquaporins are highly selective polytopic transmembrane channel proteins that facilitate the permeation of water across cellular membranes in a large diversity of organisms. Defects in aquaporin function are associated with common diseases, such as nephrogenic diabetes insipidus, congenital cataract and certain types of cancer. In general, aquaporins have a highly conserved structure; from prokaryotes to humans. The conserved structure, together with structural dynamics and the structural framework for substrate selectivity is discussed. The folding pathway of aquaporins has been a topic of several studies in recent years. These studies revealed that a conserved protein structure can be reached by following different folding pathways. Based on the available data, we suggest a complex folding pathway for aquaporins, starting from the insertion of individual helices up to the formation of the tetrameric aquaporin structure. The consequences of some known mutations in human aquaporin-encoding genes, which most likely affect the folding and stability of human aquaporins, are discussed.

The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator.

Osmolytes are naturally occurring molecules used by a wide variety of organisms to stabilize proteins under extreme conditions of temperature, salinity, hydrostatic pressure, denaturant concentration, and desiccation. The effects of the osmolyte trimethylamine N-oxide (TMAO) as well as the influence of detergent head group and acyl chain length on the stability of the Escherichia coli integral membrane protein glycerol facilitator (GF) tetramer to thermal and chemical denaturation by sodium dodecyl sulphate (SDS) are reported. TMAO promotes the association of the normally tetrameric α-helical protein into higher order oligomers in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC), as determined by dynamic light scattering (DLS); an octameric complex is particularly stable as indicated by SDS polyacrylamide gel electrophoresis. TMAO increases the heat stability of the GF tetramer an average of 10 °C in the 4 detergents and also protects the protein from denaturation by SDS. However, it did not promote re-association to the tetramer when added to SDS-dissociated protein. TMAO also promotes the formation of rod-like detergent micelles, and DLS was found to be useful for monitoring the structure of the protein and the redistribution of detergent during thermal dissociation of the protein. The protein is more thermally stable in detergents with the phosphatidylcholine head group (LLPC and LMPC) than in the maltoside detergents. The implications of the results for osmolyte mechanism, membrane protein stability, and protein-protein interactions are discussed.

Structure-activity relationships in ultrashort cationic lipopeptides: the effects of amino acid ring constraint on antibacterial activity.

Taking a minimalistic approach in efforts to lower the cost for the development of new synthetic antimicrobial peptides, ultrashort cationic lipopeptides were designed to mimic the amphiphilic nature crucial for their activity but with only a very short peptide sequence ligated to a lipidic acid. Nine ultrashort cationic lipopeptides were prepared to study the effects of ring constraint in the amino acid side chain of the peptide component. USCL-PCat1, consisting of only four L-4R-aminoproline residues and acylated with palmitic acid at the N-terminus, was found to populate a polyproline II helical secondary conformation that is stable to different pHs and temperatures using circular dichroism. The synthesized lipopeptides were found to have a micellar structure in water using negative staining transmission electron microscopy. We found that constraining the side chain of the amino acid component is not beneficial to the antimicrobial activity. USCL-Dab1, USCL-Dab3 and USCL-K1 showed promising activity against a panel of laboratory reference and clinically isolated Gram-positive and Gram-negative bacterial strains, some of which are multidrug resistant. No appreciable cytotoxicity against human monocytic THP-1 cells was observed up to concentrations of 20-40 µM for all synthesized compounds. Moreover, all USCLs did not induce the production of either pro-inflammatory cytokines or chemokines up to 40 µM.

High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

G protein-coupled receptors (GPCRs) exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM) of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP) is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯)-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(-))-TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/106 cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD) spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM.

Conformational dynamics of a membrane transport protein probed by H/D exchange and covalent labeling: the glycerol facilitator.

Glycerol facilitator (GF) is a tetrameric membrane protein responsible for the selective permeation of glycerol and water. Each of the four GF subunits forms a transmembrane channel. Every subunit consists of six helices that completely span the lipid bilayer, as well as two half-helices (TM7 and TM3). X-ray crystallography has revealed that the selectivity of GF is due to its unique amphipathic channel interior. To explore the structural dynamics of GF, we employ hydrogen/deuterium exchange (HDX) and oxidative labeling with mass spectrometry (MS). HDX-MS reveals that transmembrane helices are generally more protected than extramembrane segments, consistent with data previously obtained for other membrane proteins. Interestingly, TM7 does not follow this trend. Instead, this half-helix undergoes rapid deuteration, indicative of a highly dynamic local structure. The oxidative labeling behavior of most GF residues is consistent with the static crystal structure. However, the side chains of C134 and M237 undergo labeling although they should be inaccessible according to the X-ray structure. In agreement with our HDX-MS data, this observation attests to the fact that TM7 is only marginally stable. We propose that the highly mobile nature of TM7 aids in the efficient diffusion of guest molecules through the channel ("molecular lubrication"). In the absence of such dynamics, host-guest molecular recognition would favor semipermanent binding of molecules inside the channel, thereby impeding transport. The current work highlights the complementary nature of HDX, covalent labeling, and X-ray crystallography for the characterization of membrane proteins.

Tat peptide-calmodulin binding studies and bioinformatics of HIV-1 protein-calmodulin interactions.

The human immunodeficiency virus type 1 (HIV-1) genome encodes 18 proteins and 2 peptides. Four of these proteins encode high-affinity calmodulin-binding sites for which direct interactions with calmodulin have already been described. In this study, the HIV-1 proteome is queried using an algorithm that predicts calmodulin-binding sites revealing seven new putative calmodulin-binding sites including residues 34-56 of the transactivator of transcription (Tat). Tat is a 101-residue intrinsically disordered RNA-binding protein that plays a central role in the regulation of HIV-1 replication. Interactions between a Tat peptide (residues 34-56), melittin, a well-characterized calmodulin-binding peptide, and calmodulin were examined by direct binding studies, mass spectrometry, and fluorescence. The Tat peptide binds to both calcium-saturated and apo-calmodulin with a low micromolar affinity. Conformational changes induced in the Tat peptide were determined by circular dichroism, and residues in calmodulin that interact with the peptide were identified by HSQC NMR spectroscopy. Multiple interactions between HIV-1 proteins and calmodulin, a highly promiscuous signal transduction hub protein, may be an important mechanism by which the virus controls cell physiology.

Intrinsic disorder and function of the HIV-1 Tat protein.

The type 1 Human Immunodeficiency Virus transcriptional regulator Tat is a small RNA-binding protein essential for viral gene expression and replication. The protein binds to a large number of proteins within infected cells and non-infected cells, and has been demonstrated to impact a wide variety of cellular activities. Early circular dichroism studies showed a lack of regular secondary structure in the protein whereas proton NMR studies suggested several different conformations. Multinuclear NMR structure and dynamics analysis indicates that the reduced protein is intrinsically disordered with a predominantly extended conformation at pH 4. Multiple resonances for several atoms suggest the existence of multiple local conformers in rapid equilibrium. An X-ray diffraction structure of equine Tat, in a complex with its cognate RNA and cyclin T1, supports this conclusion. Intrinsic disorder explains the protein's capacity to interact with multiple partners and effect multiple biological functions; the large buried surface in the X-ray diffraction structure illustrates how a disordered protein can have a high affinity and high specificity for its partners and how disordered Tat assembles a protein complex to enhance transcription elongation.

High yield expression and purification of HIV-1 Tat1-72 for structural studies.

The HIV-1 transactivator of transcription (Tat) is a protein essential for virus replication. Tat is an intrinsically disordered RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven Cys residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of 6xHistidine-tagged and isotopically enriched (in N15 and C13) recombinant HIV-1 Tat(1-72) (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the backbone NMR resonances. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced, monomeric, and unfolded in aqueous solution at pH 4.

Alamethicin topology in phospholipid membranes by oriented solid-state NMR and EPR spectroscopies: a comparison.

Alamethicin, a hydrophobic peptide that is considered a paradigm for membrane channel formation, was uniformly labeled with 15N, reconstituted into oriented phosphatidylcholine bilayers at concentrations of 1 or 5 mol %, and investigated by solid-state NMR spectroscopy as a function of temperature. Whereas the peptide adopts a transmembrane alignment in POPC bilayers at all temperatures investigated, it switches from a transmembrane to an in-plane orientation in DPPC membranes when passing the phase transition temperature. This behavior can be explained by an increase in membrane hydrophobic thickness and the resulting hydrophobic mismatch condition. Having established the membrane topology of alamethicin at temperatures above and below the phase transition, ESEEM EPR was used to investigate the water accessibility of alamethicin synthetic analogues carrying the electron spin label TOAC residue at one of positions 1, 8, or 16. Whereas in the transmembrane alignment the labels at positions 8 and 16 are screened from the water phase, this is only the case for the latter position when adopting an orientation parallel to the surface. By comparing the EPR and solid-state NMR data of membrane-associated alamethicin it becomes obvious that the TOAC spin labels and the cryo-temperatures required for EPR spectroscopy have less of an effect on the alamethicin-POPC interactions when compared to DPPC. Finally, at P/L ratios of 1/100, spectral line broadening due to spin-spin interactions and thereby peptide oligomerization within the membrane were detected for transmembrane alamethicin.

Urea as a protein destabilizing agent in electrospray ionisation.

Urea is well known as a denaturant of proteins, but there is also evidence that millimolar amounts of urea may in fact stabilize protein complexes. Advances in mass spectrometric analysis have given us the opportunity to test the effect of urea on several noncovalent complexes in buffered solutions. We expected to see lower charge states if folded proteins were more compact (and therefore more stable), and higher charge states if the proteins were denatured. We have found that mM urea interferes with some noncovalent interactions, and that the extent of interference depends on the specific protein complex. The difference seems to be related to the type of interactions, with weak ones, such as H-bonds, more sensitive to urea. Examples show that a quick check with urea may give some insights into protein stability in the mass spectrometer.

Structure and alignment of the membrane-associated peptaibols ampullosporin A and alamethicin by oriented 15N and 31P solid-state NMR spectroscopy.

Ampullosporin A and alamethicin are two members of the peptaibol family of antimicrobial peptides. These compounds are produced by fungi and are characterized by a high content of hydrophobic amino acids, and in particular the alpha-tetrasubstituted amino acid residue ?-aminoisobutyric acid. Here ampullosporin A and alamethicin were uniformly labeled with (15)N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled (15)N and (31)P solid-state NMR spectroscopy. Whereas alamethicin (20 amino acid residues) adopts transmembrane alignments in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes the much shorter ampullosporin A (15 residues) exhibits comparable configurations only in thin membranes. In contrast the latter compound is oriented parallel to the membrane surface in 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine and POPC bilayers indicating that hydrophobic mismatch has a decisive effect on the membrane topology of these peptides. Two-dimensional (15)N chemical shift -(1)H-(15)N dipolar coupling solid-state NMR correlation spectroscopy suggests that in their transmembrane configuration both peptides adopt mixed alpha-/3(10)-helical structures which can be explained by the restraints imposed by the membranes and the bulky alpha-aminoisobutyric acid residues. The (15)N solid-state NMR spectra also provide detailed information on the helical tilt angles. The results are discussed with regard to the antimicrobial activities of the peptides.

The influence of sterols on the conformation of recombinant mitochondrial porin in detergent.

Mitochondrial porins (voltage-dependent anion-selective channels, VDAC) are key contributors to cellular metabolism. When isolated from mitochondria porins copurify with sterols, and some isolated forms of the protein require sterol for insertion into artificial membranes. Nonetheless, the contributions of sterols to the folded state of mitochondrial porin are not understood. Recently, with the goal of high-resolution structural studies, several laboratories have developed methods for folding recombinant porins at high concentration in detergent. In the present study, recombinant Neurospora crassa porin solubilized in detergent-sterol mixtures was examined. Sterols do not significantly alter the secondary structure of porin in lauryl dimethylamine oxide, nor in a mixture of sodium dodecylsulfate and dodecylmaltopyranoside. However, as detected by near-UV circular dichroism spectropolarimetry and fluorescence spectroscopy, the environments surrounding the aromatic amino acids in the detergent-sterol solubilized protein are measurably different from those in detergent alone. Furthermore, the effects are different in the presence of ergosterol, the native sterol in fungal mitochondria, and cholesterol. While these influences on the tertiary arrangement of detergent-solubilized porin are subtle, they may contribute to the generation of a form of the protein competent for insertion into the artificial bilayers used for electrophysiological analyses, and should be considered in future structural studies of porin.

Stability of the glycerol facilitator in detergent solutions.

Understanding membrane protein folding and stability is required for a molecular explanation of function and for the development of interventions in membrane protein folding diseases. Stable aqueous detergent solutions of the Escherichia coli glycerol facilitator in its native oligomeric state have been difficult to prepare as the protein readily unfolds and forms nonspecific aggregates. Here, we report a study of the structure and stability of the glycerol facilitator in several detergent solutions by Blue Native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD), and fluorescence. Protein tetramers were prepared in neutral dodecyl maltoside (DDM) and in zwitterionic lysomyristoylphosphatidylcholine (LMPC) detergent solutions that are stable during SDS-PAGE. Thermal unfolding experiments show that the protein is more stable in LMPC than in DDM. Tertiary structure unfolds before quaternary and some secondary structure in LMPC, whereas unfolding is more cooperative in DDM. The high stability of the protein in DDM is evident from the unfolding half-life of 8 days in 8 M urea, suggesting that hydrophobic interactions contribute to the stability. The protein unfolds readily in LMPC below pH 6, whereas the tetramer remains intact at pH 4 in DDM. At pH 4 in DDM, the protein is more sensitive than at neutral pH to unfolding by SDS and the effect is reversible. At pH 3 in DDM, the tetramer unfolds, losing its tertiary structure but retaining native helical structure which melts at significantly lower temperatures than in the native tetramer. The glycerol facilitator prepared in SDS is mainly monomeric and has ~10% less alpha-helix than the native protein. CD suggests that it forms a condensed structure with non-native tertiary contacts highly similar to the state observed in LMPC at low pH. The implications of the results for in vitro and in vivo folding of the protein are discussed.