Kerney Jebrell Glover

Structural Evaluation of Phospholipid Bicelles for Solution-State Studies of Membrane-Associated Biomolecules

Several complementary physical techniques have been used to characterize the aggregate structures formed in solutions containing dimyristoylphosphatidylcholine (DMPC)/dihexanoylphosphatidylcholine (DHPC) at ratios of < or =0.5 and to establish their morphology and lipid organization as that of bicelles. (31)P NMR studies showed that the DMPC and DHPC components were highly segregated over a wide range of DMPC/DHPC ratios (q = 0.05-0.5) and temperatures (15 degrees C and 37 degrees C). Only at phospholipid concentrations below 130 mM did the bicelles appear to undergo a change in morphology. These results were corroborated by fluorescence data, which demonstrated the inverse dependence of bicelle size on phospholipid concentration as well as a distinctive change in phospholipid arrangement at low concentrations. In addition, dynamic light scattering and electron microscopy studies supported the hypothesis that the bicellar phospholipid aggregates are disk-shaped. The radius of the planar domain of the disk was found to be directly proportional to the ratio of DMPC/DHPC and inversely proportional to the total phospholipid concentration when the DMPC/DHPC ratio was held constant at 0.5. Taken together, these results suggest that bicelles with low q retain the morphology and bilayer organization typical of their liquid-crystalline counterparts, making them useful membrane mimetics.

Lag Periods During the Self-Assembly of {Mo72Fe30} Macroions: Connection to the Virus Capsid Formation Process

The kinetic properties of the self-assembly of hydrophilic Keplerate-type polyoxometalate (POM) {Mo(72)Fe(30)} macroanions into single-layer, vesicle-like blackberry structures in solutions were monitored by the static and dynamic laser light scattering techniques. In the presence of additional electrolytes, an obvious lag period at the initial stage of self-assembly was observed, followed by a fast increase of the scattered intensity. The whole kinetic curve is sigmoidal with a lag phase. A two-step nucleation-growth mechanism is proposed to explain this lag phase: the {Mo(72)Fe(30)} macroanions slowly associate into oligomers (mostly dimers), which are the thermodynamically unfavorable intermediates, at the initial stage; once the oligomers reach a critical concentration, the blackberry formation process is accelerated. Analytical ultracentrifugation (AUC) was used to confirm the oligomeric state in {Mo(72)Fe(30)} solution during the lag period. The length of the lag period is dependent on temperature, ionic strength, and the valent states of the additional salts, as well as the solvent content. The kinetics (including the lag period) of the blackberry formation of the {Mo(72)Fe(30)} macroanions show similarities to the self-assembly of virus capsid proteins (which are also soluble macroions) into spherical capsid shells, suggesting possible connections between the self-assembly behaviors of inorganic species and biological macromolecules.

Methods for studying transmembrane peptides in bicelles: consequences of hydrophobic mismatch and peptide sequence.

We have shown that bicelles prepared from dilauryl phosphatidylcholine (DLPC) and dipalmitoyl phosphatidylcholine (DPPC) align in a magnetic field under conditions similar to the more common dimyristoyl phosphatidylcholine (DMPC) bicelles. In addition, a model transmembrane peptide, P16, with a hydrophobic stretch of 24 A, and specific alanine-d(3) labels, was incorporated into all of the different bicelles. The long-chain phospholipid (DLPC, DMPC, or DPPC) remained unperturbed upon incorporation of the peptide while the quadrupolar splitting of the short-chain phospholipid along the bicelle rim increased by varying degrees in the different bicelle systems. The change in quadrupolar splitting of the short-chain phospholipids was attributed to changes in either fluidity of the planar region of the bicelle or differences in overall lipid packing. When the hydrophobic stretch of the bilayer was 22.8 (DMPC) or 26.3 A (DPPC), the peptide tilt was found to be transmembrane (33-35 degrees with respect to the bicelle normal). When the hydrophobic stretch of the bilayer was 19.5 A (DLPC), the peptide quadrupolar splittings suggested a loss of transmembrane orientation. When tryptophan was incorporated in the middle of the transmembrane region, the transmembrane orientation was also lost.

The transmembrane domain of caveolin-1 exhibits a helix-break-helix structure.

Caveolin is an integral membrane protein that is found in high abundance in caveolae. Both the N- and C- termini lie on the same side of the membrane, and the transmembrane domain has been postulated to form an unusual intra-membrane horseshoe configuration. To probe the structure of the transmembrane domain, we have prepared a construct of caveolin-1 that encompasses residues 96-136 (the entire intact transmembrane domain). Caveolin-1(96-136) was over-expressed and isotopically labeled in E. coli, purified to homogeneity, and incorporated into lyso-myristoylphosphatidylglycerol micelles. Circular dichroism and NMR spectroscopy reveal that the transmembrane domain of caveolin-1 is primarily α-helical (57-65%). Furthermore, chemical shift indexing reveals that the transmembrane domain has a helix-break-helix structure which could be critical for the formation of the intra-membrane horseshoe conformation predicted for caveolin-1. The break in the helix spans residues 108 to 110, and alanine scanning mutagenesis was carried out to probe the structural significance of these residues. Our results indicate that mutation of glycine 108 to alanine does not disrupt the structure, but mutation of isoleucine 109 and proline 110 to alanine dramatically alters the helix-break-helix structure. To explore the structural determinants further, additional mutagenesis was performed. Glycine 108 can be substituted with other small side chain amino acids (i.e. alanine), leucine 109 can be substituted with other β-branched amino acids (i.e. valine), and proline 110 cannot be substituted without disrupting the helix-break-helix structure.

Probing the U-shaped conformation of caveolin-1 in a bilayer.

Caveolin induces membrane curvature and drives the formation of caveolae that participate in many crucial cell functions such as endocytosis. The central portion of caveolin-1 contains two helices (H1 and H2) connected by a three-residue break with both N- and C-termini exposed to the cytoplasm. Although a U-shaped configuration is assumed based on its inaccessibility by extracellular matrix probes, caveolin structure in a bilayer remains elusive. This work aims to characterize the structure and dynamics of caveolin-1 (D82-S136; Cav182-136) in a DMPC bilayer using NMR, fluorescence emission measurements, and molecular dynamics simulations. The secondary structure of Cav182-136 from NMR chemical shift indexing analysis serves as a guideline for generating initial structural models. Fifty independent molecular dynamics simulations (100 ns each) are performed to identify its favorable conformation and orientation in the bilayer. A representative configuration was chosen from these multiple simulations and simulated for 1 μs to further explore its stability and dynamics. The results of these simulations mirror those from the tryptophan fluorescence measurements (i.e., Cav182-136 insertion depth in the bilayer), corroborate that Cav182-136 inserts in the membrane with U-shaped conformations, and show that the angle between H1 and H2 ranges from 35 to 69°, and the tilt angle of Cav182-136 is 27 ± 6°. The simulations also reveal that specific faces of H1 and H2 prefer to interact with each other and with lipid molecules, and these interactions stabilize the U-shaped conformation.

Reliable expression and purification of highly insoluble transmembrane domains.

A general procedure for the reliable preparation of insoluble transmembrane domains has been developed. Improved expression schemes were developed by expressing the transmembrane domains of caveolin proteins 1, 2, and 3 as a fusion to the Trp leader protein. This construct readily formed inclusion bodies during overexpression, allowing high levels of protein to be achieved. Cleavage of the transmembrane domain away from the Trp leader carrier protein was performed with cyanogen bromide. The transmembrane domains were then purified using reverse-phase high-performance liquid chromatography with a C4 column and were eluted with a mixture of 1-butanol and acetic acid. Using this method, the 39-42 amino acid transmembrane domains from caveolin proteins 1, 2, and 3 were successfully purified to homogeneity. Further verification of this method was successfully done with Rfbp(18-51), another insoluble transmembrane domain.

Probing the caveolin-1 P132L mutant: critical insights into its oligomeric behavior and structure.

Caveolin-1 is the most important protein found in caveolae, which are cell surface invaginations of the plasma membrane that act as signaling platforms. A single point mutation in the transmembrane domain of caveolin-1 (proline 132 to leucine) has deleterious effects on caveolae formation in vivo and has been implicated in various disease states, particularly aggressive breast cancers. Using a combination of gel filtration chromatography and analytical ultracentrifugation, we found that a fully functional construct of caveolin-1 (Cav1(62-178)) was a monomer in dodecylphosphocholine micelles. In contrast, the P132L mutant of Cav1(62-178) was dimeric. To explore the dimerization of the P132L mutant further, various truncated constructs (Cav1(82-178), Cav1(96-178), Cav1(62-136), Cav1(82-136), Cav1(96-136)) were prepared which revealed that oligomerization occurs in the transmembrane domain (residues 96-136) of caveolin-1. To characterize the mutant structurally, solution-state NMR experiments in lyso-myristoylphosphatidylglycerol were undertaken of the Cav1(96-136) P132L mutant. Chemical shift analysis revealed that, compared to the wild-type, helix 2 in the transmembrane domain was lengthened by four residues (wild-type, residues 111-129; mutant, residues 111-133), which corresponds to an extra turn in helix 2 of the mutant. Lastly, point mutations at position 132 of Cav1(62-178) (P132A, P132I, P132V, P132G, P132W, P132F) revealed that no other hydrophobic amino acid can preserve the monomeric state of Cav1(62-178), which indicates that proline 132 is critical in supporting proper caveolin-1 behavior.

Recent progress in the topology, structure, and oligomerization of caveolin: a building block of caveolae.

Caveolae are cholesterol-rich plasma membrane invaginations that are found in a plethora of cell types. They play many roles including signal transduction, endocytosis, and mechanoprotection. The most critical protein in caveolae is the integral membrane protein, caveolin, which has been shown to be necessary for caveolae formation, and governs the major functions attributed to caveolae. Caveolin is postulated to act as a scaffold in the high molecular weight striated coat that surrounds the caveolar bulb, stabilizing it. Caveolin interacts, both directly and indirectly, with a large number of signaling molecules, and presides over the endocytosis of molecular cargo by caveolae. However, many of the key biophysical aspects of the caveolin protein, its structure, topology, and oligomeric behavior, are just beginning to come to light. Herein is an up-to-date summary and critique of the progress that has been made in understanding caveolin on a molecular and atomic level.

Discoid Bicelles as Efficient Templates for Pillared Lamellar Periodic Mesoporous Silicas at pH 7 and Ultrafast Reaction Times

We report the first synthesis of periodic mesoporous silicas templated by bicelles. The obtained materials form novel pillared lamellar structures with a high degree of periodic order, narrow pore size distributions, and exceptionally high surface areas.

Secondary Structure Analysis of a Functional Construct of Caveolin-1 Reveals a Long C-Terminal Helix.

Caveolin-1 is an integral membrane protein that is the primary component of cell membrane invaginations called caveolae. While caveolin-1 is known to participate in a myriad of vital cellular processes, structural data on caveolin-1 of any kind is severely limited. In order to rectify this dearth, secondary structure analysis of a functional construct of caveolin-1, containing the intact C-terminal domain, was performed using NMR spectroscopy in lyso-myristoylphosphatidylglycerol micelles. Complete backbone assignments of caveolin-1 (residues 62-178) were made, and it was determined that residues 62-79 were dynamic; residues 89-107, 111-128, and 132-175 were helical; and residues 80-88, 108-110, and 129-131 represent unstructured breaks between the helices.