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Lipoprotein signal peptidase (LspA) is an aspartyl protease that cleaves the transmembrane helix signal peptide of lipoproteins as part of the lipoprotein-processing pathway. Members of this pathway are excellent targets for the development of antibiotic therapeutics because they are essential in Gram-negative bacteria, are important for virulence in Gram-positive bacteria, and may not develop antibiotic resistance. Here, we report the conformational dynamics of LspA in the apo state and bound to the antibiotic globomycin determined using molecular dynamics simulations and electron paramagnetic resonance. The periplasmic helix fluctuates on the nanosecond timescale and samples unique conformations in the different states. In the apo state, the dominant conformation is the most closed and occludes the charged active site from the lipid bilayer. With antibiotic bound there are multiple binding modes with the dominant conformation of the periplasmic helix in a more open conformation. The different conformations observed in both bound and apo states indicate a flexible and adaptable active site, which explains how LspA accommodates and processes such a variety of substrates.

Currently, membrane proteins only comprise 1.5% of the protein data bank and, thus, still remain a challenge for structural biologists. Expression, stabilization in membrane mimics (e.g. detergent), heterogeneity (conformational and chemical), and crystallization in the presence of a membrane mimic are four major bottlenecks encountered. In response, several post-expression protein modifications have been utilized to facilitate structure determination of membrane proteins. This review highlights four approaches: limited proteolysis, deglycosylation, cysteine alkylation, and lysine methylation. Combined these approaches have facilitated the structure determination of more than 40 membrane proteins and, therefore, are a useful addition to the membrane protein structural biologist's toolkit.

Hemoglobin has been studied and well haracterized in red blood cells for over one hundred years. However, new work has indicated that the hemoglobin alpha subunit (Hbα) is also found within the blood vessel wall, where it appears to localize at the myoendothelial junction (MEJ) and plays a role in regulating nitric oxide (NO) signaling between endothelium and smooth muscle. This discovery has created a new paradigm for control of endothelial nitric oxide synthase activity, nitric oxide diffusion, and ultimately, control of vascular tone and blood pressure. This review will discuss the current knowledge of hemoglobin’s properties as a gas exchange molecule in the blood stream, and extrapolate the properties of Hbα biology to the MEJ signaling domain. Specifically, we propose that Hbα is present at the MEJ to regulate NO release and diffusion in a restricted physical space, which would have powerful implications for the regulation of blood flow in peripheral resistance arteries.

Carcinoembryonic antigen-like cell adhesion molecules (CEACAMs) are human cell-surface proteins that can exhibit increased expression on tumor cells, and are thus a potential target for novel tumor-seeking therapeutic delivery methods. We hypothesize that engineered nanoparticles containing a known interaction partner of CEACAM, the Neisseria gonorrhoeae outer membrane protein Opa, can be used to deliver cargo to specific cellular targets. In this study, the cell association and uptake of protein-less liposomes and Opa proteoliposomes into CEACAM-expressing cells was measured using imaging flow cytometry. A size-dependent internalization of liposomes into HeLa cells was observed through endocytic pathways. Opa-dependent, CEACAM1-mediated uptake of liposomes into HeLa cells was observed, with very little colocalization with endosomal and lysosomal trafficking compartments. Given the overexpression of CEACAM1 on several distinct cancers and the strong interest in using CEACAM1 as a component in treatment strategies, these results support further pursuit of investigating Opa-dependent specificity and internalization mechanism for therapeutic delivery.

Nitric oxide (NO), an endogenously produced gaseous molecule, is the most potent way of regulating blood pressure in the resistance vasculature. NO is produced in endothelial cells by an oxidoreductase enzyme, endothelial nitric oxide synthase (eNOS), and diffuses to the surrounding smooth muscle cells to cause relaxation and vasodilation. As part of the cardiovascular system, eNOS is subject to tight control to maintain blood pressure homeostasis. A recently characterized inhibitor of NO signaling, the alpha subunit of hemoglobin (alpha globin) is expressed in endothelial cells and acts both as an NO scavenger and a direct inhibitor of NO production via interaction with eNOS. Using an alpha globin mimetic peptide (Hbα-X), we seek to understand the molecular determinants of alpha globin's inhibitory binding properties. Using fluorescence polarization, we find a low nanomolar affinity for the interaction between the oxygenase domain of eNOS and Hbα-X. Biolayer interferometry has been used to verify this result. Application of Hbα-X to humans found a significant inhibition of arterial reactivity in hypertensive individuals. In vivo administration of the peptide shows a significant decrease in mean arterial pressure in mice, which could be explained by competition for binding regions with the endogenous alpha globin. Understanding the interaction between alpha globin and eNOS is the first step towards rational design of a peptide with greater affinity for therapeutic use in hypertension.

Neisseria gonorrhoeae is a unique pathogen in that it not only colonizes human tissue by interactions with host receptors, but these interactions can trigger engulfment of the bacteria even by typically non-phagocytic cells. N. gonorrhoeae possess a family of opacity-associated (Opa) proteins, which are 8-stranded β-barrel outer membrane proteins that bind to human host cell receptors. The majority of Opa proteins engage select human CEACAM receptors (carcino-embryonic antigen-like cellular adhesion molecules), and this interaction can trigger phagocytosis of the bacterium. To date, there are 345 distinct opa alleles sequenced; the most prominent differences are in the second and third extracellular loops (hypervariable regions, HV1 and HV2). In addition to contributing to the bacteria's ability to evade the human immune system, these HV regions determine receptor specificity. While the Opa protein family has conserved structural elements, the molecular determinants of receptor recognition and engagement remain unclear. We aim to compare the interactions of various Opa proteins in vitro (reconstituted into liposomes) in order to define how differences in Opa sequence and structure inform receptor binding. To investigate structural motifs involved in Opa-receptor interactions, we are conducting binding assays and protein footprinting experiments to determine how differences in sequence affect receptor engagement. Affinities for a selection of Opa proteins with a variety of CEACAM receptors were determined using biolayer interferometry, with KD values in the low nM range. Protease protection assays are currently being performed to determine which amino acid residues in the HV regions are involved in receptor recognition and engagement. Together, the results of these experiments provide insight into the molecular determinants of the Opa-CEACAM interaction, and provide a clearer understanding of the bacterial mechanism for engulfment into human host cells.

The pathogenic bacteria Neisseria gonorrhoeae and N. meningitidis induce their own phagocytosis into human host cells prior to replication. Bacterial cell entry is promoted by the binding of Neisserial outer membrane opacity-associated (Opa) proteins to human carcinoembryonic antigen-like cell adhesion molecule (CEACAM) receptors, a widely-distributed class of cell receptors in the human body. Binding of Opa proteins to either CEACAM1, CEACAM3, CEACAM5, or CEACAM6 generates intracellular signalling events which lead to bacterial internalization by both phagocytes and epithelial cells. We are interested in determining whether purified and folded Opa proteins stabilized in liposomes retain the ability to promote liposomal entry into epithelial cells similar to Neisseria. The ability to target CEACAM+ cells for Opa proteoliposome uptake may prove valuable in targeted therapeutic delivery. Our results demonstrate that Opa-proteoliposomes target surface CEACAM receptors on transfected HeLa cells for binding and preliminary data indicate the liposomes are taken into the cells. This internalization is dependent on metabolic energy, suggesting the cells may employ active-uptake mechanisms to internalize the liposomes. Our work shows that Opa proteoliposomes deserve further consideration as a platform for targeted delivery to CEACAM-expressing cells.

Micelles and bicelles are used to isolate membrane proteins for biophysical characterization, functional assays, and high-resolution structure determination. Utilizing these membrane mimics requires an extensive screening process to identify the appropriate amphiphilic environment to stabilize a functional membrane protein. To enable rational mimic selection, the goal of this research is to investigate the structural properties of micelles and bicelles to determine how their physical characteristics can forward membrane protein research. Small angle X-ray scattering and small angle neutron scattering were used to investigate the structure of micelles and bicelles, specifically binary mixtures of detergents and lipids. To determine the impact of membrane mimic structure on protein function, outer membrane phospholipase A1, the protease OmpT, and the lipid A palmitoyltransferase PagP were purified in detergent micelles with varying properties, and the overall protein activity was evaluated. Hypotheses based on the correlations between activity and micelle properties were tested using mixed micelles and bicelles with tunable properties. The information gained from these studies will determine the physical properties important in stabilizing membrane proteins and provide guiding principles for selecting membrane mimics.

Detergent micelles are often used as a bilayer mimic for membrane proteins for functional assays and high-resolution structural studies, but selecting the appropriate detergent for the protein requires extensive time and resources. The long-term goal of this research is to rationally select a membrane mimic for a given membrane protein that stabilizes fold and native function based on matching physical properties of the detergent and protein. To determine the important micelle and membrane properties, lipoprotein signal peptidase A (LspA) from Chlamydia trachomatis is used as a model system. To screen detergents that support the native function, LspA was purified in over 30 detergent micelles with varying physical properties. LspA is soluble in zwitterionic detergents and solubility remains independent of detergent alkyl chain length, solubilizing in detergents with wide range of hydrophobic radius, 27-37A. A detergent may maintain protein solubility without maintaining function or fold; therefore, the function of LspA in these detergents is currently being studied. To assess the activity of native membrane and solubilized LspA, a signal peptide containing the LspA cleavage consensus sequence and donor-quencher pair on each termini was designed. Preliminary HPLC and fluorescence data demonstrate a cleavage of the signal peptide upon addition of LspA in the native membrane and a comparison to different protein-detergent complexes will be presented. The results presented will identify solubilizing detergents, monitor enzymatic activity, and ultimately correlate the physical properties of the solubilizing detergent and protein structure and function.

Flexible proteins present a unique set of challenges for refinement with either computational or experimental techniques: because these proteins occupy a large conformational space, standard molecular dynamics (MD) simulations can be prohibitively expensive, and high-resolution experimental techniques like x-ray crystallography intentionally capture only small set of low energy states. A new approach is therefore needed to capture the conformational ensembles of flexible proteins. Double electron-electron resonance (DEER) spectroscopy, an experimental technique that measures distance distributions between pairs of spin-labeled residues on a protein, provides quantitative information about less populated but still potentially important conformational states. However, because DEER experiments are costly and time consuming, only a small subset of pairs can be measured, often chosen arbitrarily. If instead of selecting an arbitrary or random subset, we select a subset of pairs such that they provide the maximum amount of information about the conformational ensemble, DEER could become an even more powerful tool for refinement. We have therefore developed 1) a new information-theoretic metric that ranks pairs of residues based on how well they refine a conformational ensemble and 2) a new methodology which identifies a set of highly informative pairs that perform well under this metric. Specifically, we incorporate DEER distance distributions into restrained-ensemble MD simulations, utilize the data from these simulations to identify a set of maximally-informative and minimally-redundant pairs, measure the pairs with DEER, and demonstrate the use of these pairs in refining the conformational ensemble of a flexible protein.

Micelle-forming detergents are used to solubilize integral membrane proteins for biochemical and physical characterizations. However, the unique properties of each membrane protein require exhaustive, and currently empirical, screening to optimize the detergent conditions which yield a stable protein-detergent complex (PDC). Detergent mixtures provide a means of expanding the available micellar environments, while also allowing select properties of micelles to be engineered. The properties of detergent mixtures must be well understood to correlate these properties with the stability of the protein fold and function. Using small-angle X-ray scattering, we determined the sizes and shapes of micelles formed by a comprehensive set of commercially available detergents commonly used with membrane proteins, and systematically assessed binary mixtures of these detergents. Micelle size and shape were determined directly from a Guinier analysis of the low angle data, the position of the second maxima at intermediate angles, and a core-shell model fit to the micelle scattering profiles. Many micelle properties, such as hydrophobic thickness, have a linear dependence on the micelle mole fraction. In addition to modulating the size of the micelle, other properties such as surface charge and fluidity can also be engineered. The results of this investigation can now be used to rationally design micelles for membrane protein investigations.

Pathogenic Neisseria gonorrhoeae, the causative agent of gonorrhea, possess a family of outer membrane proteins referred to as opacity-associated (Opa) proteins. These Opa proteins are β-barrel outer membrane proteins that bind to human host cell receptors, inducing engulfment of the bacterium. To date, there have been over 300 distinct opa alleles sequenced. The differences in sequence have stemmed primarily from recombination events, and are most pronounced in two regions of the protein in the second and third extracellular loops (termed hypervariable regions, HV1 and HV2). These HV regions are responsible for determining receptor specificity. The most abundant Opa family engages human CEACAM receptors (carcino-embryonic antigen-like cellular adhesion molecules). While the Opa protein family has conserved structural elements, the molecular determinants of the receptor interactions are unknown. We aim to compare the function of a variety of Opa proteins reconstituted into liposomes in order to define how differences in Opa sequence and structure determine receptor binding. To investigate the structural motif for Opa-receptor recognition and engagement, we are employing techniques that will yield both low and high resolution models of the Opa-receptor complex. Affinities for a variety of CEACAM-binding Opa family members were determined using fluorescence polarization, with dissociation constant values ranging from ∼1-10 nM. To determine which specific amino acids are involved in receptor recognition, competition assays were performed using different peptides that mimic segments of the HV regions of Opa proteins. X-ray crystallography of the Opa-CEACAM complex is being pursued for a higher resolution understanding, and progress towards the structure will be presented. The results of these experiments provide insights into the specific molecular determinants of Opa protein interactions with host receptors.

We have developed a combined experimental and computational approach for understanding the conformational equilibria of opacity associated adhesin (Opa) protein binding to human carcinoembryonic cell adhesin molecule 1 (CEACAM). This binding is a flexible recognition process: although the Opa-CEACAM binding event is high-affinity and shows some specificity, the unliganded state of Opa is extremely flexible, and Opa is highly tolerant of mutations that still permit CEACAM recognition. To elucidate the nature of this binding process, we combine electron paramagnetic resonance experiments and molecular dynamics simulation as follows. Double electron electron resonance (DEER) distance measurements between Opa and CEACAM residues are used to inform molecular dynamics simulations of the Opa-CEACAM complex. We incorporate DEER measurements by adding spring potentials to the system Hamiltonian. In order to thoroughly sample the system's free energy landscape, we use a MARTINI coarse-grained model, cluster the resultant trajectories, and begin additional atomistic simulations from cluster centers. The benefits of this approach are twofold. First, we can direct DEER experiments by identifying under-determined regions of phase space. We identify these regions via a maximum-information-minimum-redundancy approach: the mutual information is computed between each pair Opa-CEACAM residue pair and the system's configuration, and then highly informative pairs are measured using DEER. Second, once the model is validated by DEER, we can use the same clustering methods to identify which conformational states and specific interactions contribute to Opa-CEACAM binding.

Neisseria gonorrhoeae and N. meningitides are human pathogens which infect millions each year and are becoming increasingly antibiotic resistant. These bacteria utilize opacity-associated (Opa) proteins to mediate bacterial uptake into non-phagocytic and phagocytic cells as a means of infection. Opa proteins primarily engage carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs), hijacking host cellular mechanisms to induce bacterial engulfment. The Opa family of proteins are outer membrane β-barrels with eight transmembrane strands and four extracellular loops. Opa proteins bind CEACAMs with high affinity and specificity, yet the loops important to receptor engagement are highly diverse in sequence among Opa variants. We aim to determine how sequence diversity in the receptor-interacting domains of Opa loops facilitates engagement of CEACAM receptors. Results from CW-EPR and NMR relaxation experiments indicate the Opa loops are dynamic on the nanosecond timescale in lipid and detergent environments. Such flexibility may enable sampling of transient structures and interactions that promote receptor engagement. To determine if the conformation of the bound loops is sampled, the structure of the Opa-CEACAM complex is needed. Towards this end, a model of the Opa60-CEACAM1 binding complex will be determined using a hybrid Double Electron Electron Resonance (DEER) and molecular dynamics (MD) approach. DEER distances between Opa60 and CEACAM1 provide spatial restraints for MD simulations of the complex, and preliminary models will be presented. Elucidating the mechanism of Opa-receptor interactions will augment our understanding of the determinants promoting the entry of a foreign body into non-phagocytic cells.

Membrane protein research is hampered by the difficulty in selecting a membrane mimic that solubilizes and stabilizes protein fold and function. While detergents are often utilized, identifying the appropriate detergent composition to maintain protein solubilization and stability is an expensive, time-consuming, empirical process that is often unsuccessful. The goal of this research is to understand the interactions between detergent and protein by relating physical surfactant properties with membrane protein fold, function, and stability, enabling rational detergent selection. To determine important micelle and protein characteristics, outer membrane phospholipase A1 (OMPLA), the protease OmpT, and the lipid A palmitoyltransferase PagP were purified in different detergent micelles with varying properties such as alkyl chain length, charge, and head group. The overall protein structure and function were evaluated in many pure micelles to identify trends with detergent characteristics and protein function. Upon determination of kinetic parameters for several β-barrel proteins in pure micelles, protein function and structure will be investigated with mixed micelles, to test hypotheses generated by the trends observed in pure micelles. This research will provide a logical rationale for the selection of detergents based on the physical properties of membrane proteins and detergents.

One major bottleneck to the investigation of membrane proteins is the stabilization of structure and function in detergents and lipid bilayers after purification from the native membrane. Detergents are the most successful membrane mimic, thus far, for NMR and X-ray crystallography structure determination of membrane proteins. However, the current empirical screening of detergents that stabilize protein function and fold is laborious, costly, and often is not successful. To better understand the physical determinants that stabilize a protein-detergent complex, we have systematically investigated the properties of detergent micelles. Specifically, we have determined that binary detergent mixtures form ideally mixed micelles and that many physical properties are different from the pure individual micelles and vary linearly with micelle mole fraction. The predictability of the shape, size, and surface properties of binary mixtures expands the molecular toolkit for applications that utilize detergents and provide a means to systematically test the influence these properties have on membrane protein fold and function. Our progress towards correlating detergent micelle physical properties with membrane protein structure and function will be presented.

Understanding the structure and functional dynamics of membrane proteins in their native, hydrophobic environment is key to understanding how proteins function. EPR spectroscopy in combination with site directed spin labeling (SDSL) has the potential to quantify structure and dynamics of proteins of arbitrary weight in their native lipid environment. Several studies have elucidated the structural origins of CW EPR lineshapes of water-soluble proteins; however, CW EPR spectra of nitroxide spin labeled proteins in a detergent/lipid environment have characteristic differences from their water-soluble counterparts. Membrane protein spectra are generally broader and frequently contain multiple spectral components. In this study, membrane exposed sites on model α-helical and β-barrel proteins LeuT and OmpA, respectively, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations in order to obtain a motional model of the nitroxide. For each crystal structure of spin labeled LeuT, the nitroxide ring of the spin label is resolved and makes contacts with hydrophobic residues on the protein surface. In addition to the crystal structures, the spectral effect of nearest neighbor mutations and the spectral simulations suggest the spin label has a greater affinity for its local protein environment compared to water-soluble proteins. These results for spin labeled sites on LeuT and OmpA begin to explain the observed trends in membrane protein CW EPR spectra.