Alison H. Dewald

Physical Determinants of β-Barrel Membrane Protein Folding in Lipid Vesicles

The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa)(60) and Opa(50) into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.

A valveless microfluidic device for integrated solid phase extraction and polymerase chain reaction for short tandem repeat (STR) analysis

A valveless microdevice has been developed for the integration of solid phase extraction (SPE) and polymerase chain reaction (PCR) on a single chip for the short tandem repeat (STR) analysis of DNA from a biological sample. The device consists of two domains--a SPE domain filled with silica beads as a solid phase and a PCR domain with an ~500 nL reaction chamber. DNA from buccal swabs was purified and amplified using the integrated device and a full STR profile (16 loci) resulted. The 16 loci Identifiler® multiplex amplification was performed using a non-contact infrared (IR)-mediated PCR system built in-house, after syringe-driven SPE, providing an ~80-fold and 2.2-fold reduction in sample and reagent volumes consumed, respectively, as well as an ~5-fold reduction in the overall analysis time in comparison to conventional analysis. Results indicate that the SPE-PCR system can be used for many applications requiring genetic analysis, and the future addition of microchip electrophoresis (ME) to the system would allow for the complete processing of biological samples for forensic STR analysis on a single microdevice.

Known Structure, Unknown Function: An Inquiry-based Undergraduate Biochemistry Laboratory Course

Undergraduate biochemistry laboratory courses often do not provide students with an authentic research experience, particularly when the express purpose of the laboratory is purely instructional. However, an instructional laboratory course that is inquiry‐ and research‐based could simultaneously impart scientific knowledge and foster a students research expertise and confidence. We have developed a year‐long undergraduate biochemistry laboratory curriculum wherein students determine, via experiment and computation, the function of a protein of known three‐dimensional structure. The first half of the course is inquiry‐based and modular in design; students learn general biochemical techniques while gaining preparation for research experiments in the second semester. Having learned standard biochemical methods in the first semester, students independently pursue their own (original) research projects in the second semester. This new curriculum has yielded an improvement in student performance and confidence as assessed by various metrics. To disseminate teaching resources to students and instructors alike, a freely accessible Biochemistry Laboratory Education resource is available at http://biochemlab.org.

A modular microfluidic system for deoxyribonucleic acid identification by short tandem repeat analysis

Microfluidic technology has been utilized in the development of a modular system for DNA identification through STR (short tandem repeat) analysis, reducing the total analysis time from the ∼6 h required with conventional approaches to less than 3h. Results demonstrate the utilization of microfluidic devices for the purification, amplification, separation and detection of 9 loci associated with a commercially-available miniSTR amplification kit commonly used in the forensic community. First, DNA from buccal swabs purified in a microdevice was proven amplifiable for the 9 miniSTR loci via infrared (IR)-mediated PCR (polymerase chain reaction) on a microdevice. Microchip electrophoresis (ME) was then demonstrated as an effective method for the separation and detection of the chip-purified and chip-amplified DNA with results equivalent to those obtained using conventional separation methods on an ABI 310 Genetic Analyzer. The 3-chip system presented here demonstrates development of a modular, microfluidic system for STR analysis, allowing for user-discretion as to how to proceed after each process during the analysis of forensic casework samples.

Label-Free Method for Cell Counting in Crude Biological Samples via Paramagnetic Bead Aggregation

Under chaotropic conditions, DNA released from lysed cells causes the aggregation of paramagnetic beads in a rotating magnetic field in a manner that is independent of the presence of other cellular components. The extent of aggregation correlates with the mass of DNA in a quantitative manner (Leslie, D. C. et al., J. Am. Chem. Soc. 2012, 134, 5689-96), and from this, the number of DNA-containing cells in the sample can be enumerated. Microbial growth testing is demonstrated by monitoring bead aggregation with E. coli in the presence of ampicillin. Without the need for fluorescent labeling or Coulter counting, the white blood cell count can be defined directly from a microliter of crude whole blood. Specificity is brought to the process by coupling bead-based immunocapture with DNA-bead aggregation allowing for the enumeration of CD4+ T cells from human blood samples. The results of DNA-induced bead aggregation had a 95% correlation with those generated by flow cytometry. With the process requiring only inexpensive, widely available benchtop laboratory hardware, a digital camera, and a simple algorithm, this provided a highly accessible alternative to more expensive cell-counting techniques.

Neisserial Opa Protein-CEACAM Interactions: Competition for Receptors as a Means of Bacterial Invasion and Pathogenesis.

Carcino-embryonic antigen-like cellular adhesion molecules (CEACAMs), members of the immunoglobulin superfamily, are responsible for cell-cell interactions and cellular signaling events. Extracellular interactions with CEACAMs have the potential to induce phagocytosis, as is the case with pathogenic Neisseria bacteria. Pathogenic Neisseria species express opacity-associated (Opa) proteins, which interact with a subset of CEACAMs on human cells, and initiate the engulfment of the bacterium. We demonstrate that recombinant Opa proteins reconstituted into liposomes retain the ability to recognize and interact with CEACAMs in vitro but do not maintain receptor specificity compared to that of Opa proteins natively expressed by Neisseria gonorrhoeae. We report that two Opa proteins interact with CEACAMs with nanomolar affinity, and we hypothesize that this high affinity is necessary to compete with the native CEACAM homo- and heterotypic interactions in the host. Understanding the mechanisms of Opa protein-receptor recognition and engulfment enhances our understanding of Neisserial pathogenesis. Additionally, these mechanisms provide insight into how human cells that are typically nonphagocytic can utilize CEACAM receptors to internalize exogenous matter, with implications for the targeted delivery of therapeutics and development of imaging agents.

Protein and Environmental Determinants of OMP Fate in Liposomes

Despite the vast importance of outer membrane proteins (OMPs), OMPs are understudied because they must first be folded into a liposome or other membrane mimetic.1 There is no ideal folding condition that works for all OMPs, so empirical screens are used to determine the best conditions for a given OMP to fold.2 We aim to analyze the fates of various OMPs (fold, aggregate, or lipid-associate without folding) in a variety of conditions to understand the protein (particularly extracellular loop), lipid, and buffer characteristics that determine the proteins’ fate. Theoretical understanding of folding determinants would significantly decrease the time needed to find a folding system for any particular OMP, which could expedite in vitro work on membrane proteins. Four OMPs were chosen for study: OmpA, OmpW, Opa60 and OmpX. OmpA and OmpX are promiscuous folders, while Opa60 and OmpW are difficult folders.2,3 All four are eight stranded β-barrels, with extracellular loops of varying size, pI, and hydrophobicity. The OMPs were overexpressed to inclusion bodies and purified using centrifugation and wash steps, with final purity demonstrated by SDS-PAGE. OMPs were folded into liposomes (SUVs and LUVs) and protein aggregate was separated from folded and lipid-associated protein via ultra-centrifugation.3 Folded and unfolded protein in each fraction was determined by SDS-PAGE and gel densiometry, as fully folded β-barrels migrate differently than lipid-associated protein through a polyacrylamide gel. Results for the four OMPs at various pH, lipid chain length, and salt concentrations are presented.1. LK Tamm, H Hong, and B Liang. (2004) Biochim Biophys Acta, 1666, 250.2. NK Burgess, TP Dao, AM Stanley, KG Fleming. (2008) J Biol Chem, 283, 26748.3. AH Dewald, JC Hodges, L Columbus. (2011) Biophys J, 100, 2131.

The Characterization of the Binding of Opacity-Associated Proteins to Human Host Cell Receptors

Opacity-associated (Opa) proteins are eight-stranded β-barreled monomeric outer membrane proteins found in the bacterial pathogens Neisseria meningitides and N. gonorrhoeae. In vivo, these proteins interact with specific human host cell receptors to induce phagocytosis of the bacterium, allowing Neisseria to breach the plasma membrane and gain entry to targeted human cells. There are at least 26 characterized Opa proteins, nearly identical in sequence except for three extracellular loops. Host-receptor specificity is determined by the variable extracellular loops; however, the specific molecular determinants of the interaction have not been identified. Opa proteins are classified into two families based on their host receptor selectivity, the larger class, OpaCEA, bind to carcinoembryonic antigen-like cellular adhesion molecules (CEACAMs) and the smaller class, OpaHS, bind to heparansulfate proteoglycan receptors (HSPGs) or to integrin receptors via an HSPG-mediated interaction with fibronectin or vitronectin. Though the Opa-host receptor interaction is directly related to the invasion efficiency of Neisseria, the thermodynamics (e. g. affinities) of Opa proteins interactions with host receptors is not known. We present a study investigating the binding of two Opa protein variants, OpaI (an OpaCEA) and OpaA (an OpaHS) with their cognate host receptors. A centrifugal “pull-down” assay is used with fluorescence spectroscopy to determine the specificity and affinity of the Opa - receptor interactions. Although attempts have been made to study Opa specificity in vivo, this study demonstrates Opa protein selective binding to the respective receptors in vitro, thereby facilitating investigations of protein determinants relating both to binding specificity and affinity.