Daniel A. Fox

Dependence of micelle size and shape on detergent alkyl chain length and head group

Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2–1.5 Å per carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ∼16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.

Mixing and Matching Detergents for Membrane Protein NMR Structure Determination

One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and small-angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the proteins hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

Structure of the neisserial outer membrane protein opa60: loop flexibility essential to receptor recognition and bacterial engulfment.

The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen–host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors.

Solution NMR resonance assignment strategies for β-barrel membrane proteins

Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of β‐barrel membrane proteins using Opa60 from Neisseria gonorrhoeae as a model system. Opa60 is an eight‐stranded β‐barrel with long extracellular loops (∼63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa60 in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the β‐barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized β‐barrel. The removal of the loop resonances significantly improved the assignment of the Opa60 β‐barrel region (97% of the resonances corresponding to the β‐barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full‐length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of β‐barrel membrane proteins.

Tuning Micelle Dimensions and Properties with Binary Surfactant Mixtures

Detergent micelles are used in many areas of research and technology, in particular, as mimics of the cellular membranes in the purification and biochemical and structural characterization of membrane proteins. Applications of detergent micelles are often hindered by the limited set of properties of commercially available detergents. Mixtures of micelle-forming detergents provide a means to systematically obtain additional micellar properties and expand the repertoire of micelle features available; however, our understanding of the properties of detergent mixtures is still limited. In this study, the shape and size of binary mixtures of seven different detergents commonly used in molecular host-guest systems and membrane protein research were investigated. The data suggests that the detergents form ideally mixed micelles with sizes and shapes different from those of pure individual micelles. For most measurements of size, the mixtures varied linearly with detergent mole fraction and therefore can be calculated from the values of the pure detergents. We propose that properties such as the geometry, size, and surface charge can be systematically and predictably tuned for specific applications.

NMR Backbone Assignment of Opai: A Mediator of Host:Neisseria Interactions

Opa (opacity associated) proteins from Neisseria gonorrhoeae (NG) and Neisseria meningitides (NM) induce phagocytosis of the bacterium by non-phagocytic cells such as epithelial cells. There are eleven NG and four NM Opa alleles that undergo phase-variation expression to the outer membrane. The protein products are nearly identical in sequence, but vary in three extracellular loop regions that determine the host receptor specificity. Opa proteins bind to carcinoembryonic antigen-like cellular adhesion molecules (CEACAMs) or to heparansulfate proteoglycan receptors (HSPGs), thus subdividing Opa proteins into two classes, OpaCEA and OpaHS, respectively. Mutational and chimeric experiments have not revealed the sequence determinants of the hypervariable regions that are responsible for receptor recognition. The goal of this study is to investigate the structure, dynamics, and receptor interactions of OpaI, a 27 kDa, 238 amino acid OpaCEA from NG MS11. OpaI was over-expressed in E. coli, purified, and refolded in dodecylphosphocholine. Two-dimensional 15N, 1H- TROSY spectra, CD, and SDS-PAGE gel mobility of the protein-detergent complex indicate that the protein is folded and well-suited for NMR studies Using TROSY-based pulse sequences, methyl labeling, and specific amino acid labeling, a suitable backbone assignment was achieved and an initial low-resolution structural calculation with H-bond and NOE-derived restraints is presented.

Molecular Determinants of Neisserial Pathogenesis: Mapping the Interaction Between Opa I and a Human Binding Partner CEACAM1

Neisserial Opa proteins mediate the internalization of the bacterial cell by host epithelial cells via an interaction between the extracellular loops of Opa proteins and the extracellular domains of the host binding partner present on the cell surface. The eleven Opa proteins can be subdivided into two classes on the basis of the human receptor target. The OpaHS class is named for heparan sulfate proteoglycan (HSPG), while OpaCEA proteins bind carcinoembryonic-antigen related cell adhesion molecules (CEACAMs), of which there are seven varieties. Significantly, each of the OpaCEA proteins has a characteristic specificity for each CEACAM. Of the four extracellular loops of Opa proteins, binding specificity is attributed primarily to two, which correspond to hypervariable regions of the protein sequence. However, mutational and chimeric analyses have not revealed the sequence determinants of the hypervariable regions that are responsible for receptor target recognition. Furthermore, it has been shown that the binding requires a cooperative interaction between the two hypervariable domains, and that specificity is determined by specific pairing of the sequences. It is the goal of this study to determine at a molecular level how specificity is attained by studying the structure and dynamics of the Opa I - receptor interactions. To this end, OpaI, which binds to CEACAM1 receptors, has been cloned, expressed, purified, and refolded and the NMR backbone assignment is in progress. The progress towards structure determination will be presented. In addition, NMR data mapping the interactions between Opa I and the soluble receptor will be presented in order to characterize the functionally relevant structural interactions involved in bacterial pathogenesis.