Svetlana Kotova

Structure of the Plasmodium falciparum Circumsporozoite Protein, a Leading Malaria Vaccine Candidate

The Plasmodium falciparum circumsporozoite protein (CSP) is critical for sporozoite function and invasion of hepatocytes. Given its critical nature, a phase III human CSP malaria vaccine trial is ongoing. The CSP is composed of three regions as follows: an N terminus that binds heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C terminus that contains a thrombospondin-like type I repeat (TSR) domain. Despite the importance of CSP, little is known about its structure. Therefore, recombinant forms of CSP were produced by expression in both Escherichia coli (Ec) and then refolded (EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP) for structural analyses. To analyze the TSR domain of recombinant CSP, conformation-dependent monoclonal antibodies that recognized unfixed P. falciparum sporozoites and inhibited sporozoite invasion of HepG2 cells in vitro were identified. These monoclonal antibodies recognized all recombinant CSPs, indicating the recombinant CSPs contain a properly folded TSR domain structure. Characterization of both EcCSP and PpCSP by dynamic light scattering and velocity sedimentation demonstrated that both forms of CSP appeared as highly extended proteins (Rh 4.2 and 4.58 nm, respectively). Furthermore, high resolution atomic force microscopy revealed flexible, rod-like structures with a ribbon-like appearance. Using this information, we modeled the NANP repeat and TSR domain of CSP. Consistent with the biochemical and biophysical results, the repeat region formed a rod-like structure about 21–25 nm in length and 1.5 nm in width. Thus native CSP appears as a glycosylphosphatidylinositol-anchored, flexible rod-like protein on the sporozoite surface.

Site-directed mutagenesis of La protease: A catalytically active serine residue

Comparative sequence analysis of Escherichia coli ATP‐dependent La protease led to the suggestion that Ser679 is the catalytically active enzyme residue. Site‐directed mutagenesis Ser679 → Ala, investigation of the cells containing the mutant plasmid, and study of the partially purified mutant protein produced results in favour of this suggestion.

Initial Stages of V(D)J Recombination: The Organization of RAG1/2 and RSS DNA in the Postcleavage Complex

To obtain structural information on the early stages of V(D)J recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved recombination signal sequences (RSS). Stoichiometric and molecular mass analysis established that this signal-end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative-staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near the RAG1 N termini. These first images of the V(D)J recombinase in its postcleavage state provide a framework for modeling RAG domains and their interactions with DNA.

Analysis of the Conformation and Function of the Plasmodium falciparum Merozoite Proteins MTRAP and PTRAMP

ABSTRACT Thrombospondin repeat (TSR)-like domains are structures involved with cell adhesion. Plasmodium falciparum proteins containing TSR domains play crucial roles in parasite development. In particular, the preerythrocytic P. falciparum circumsporozoite protein is involved in hepatocyte invasion. The importance of these domains in two other malaria proteins, the merozoite-specific thrombospondin-related anonymous protein (MTRAP) and the thrombospondin-related apical membrane protein (PTRAMP), were assessed using near-full-length recombinant proteins composed of the extracellular domains produced in Escherichia coli. MTRAP is thought to be released from invasive organelles identified as micronemes during merozoite invasion to mediate motility and host cell invasion through an interaction with aldolase, an actin binding protein involved in the moving junction. PTRAMP function remains unknown. In this study, the conformation of recombinant MTRAP (rMTRAP) appeared to be a highly extended protein (2 nm by 33 nm, width by length, respectively), whereas rPTRAMP had a less extended structure. Using an erythrocyte binding assay, rMTRAP but not rPTRAMP bound human erythrocytes; rMTRAP binding was mediated through the TSR domain. MTRAP- and in general PTRAMP-specific antibodies failed to inhibit P. falciparum development in vitro. Altogether, MTRAP is a highly extended bifunctional protein that binds to an erythrocyte receptor and the merozoite motor.

Retinoschisin (RS1) interacts with negatively charged lipid bilayers in the presence of Ca2+: an atomic force microscopy study.

Retinoschisin (RS1) is a retina-specific secreted protein encoding a conserved discoidin domain sequence. As an adhesion molecule, RS1 preserves the retinal cell architecture and promotes visual signal transduction. In young males, loss-of-function mutations in the X-linked retinoschisis gene (RS1) cause X-linked retinoschisis, a form of progressive blindness. Neither the structure of RS1 nor the nature of its anchoring and organization on the plasma membranes is fully understood. The discoidin C2 domains of coagulation factors V and VIII are known to interact with extracellular phosphatidylserine (PS). In this study we have used atomic force microscopy (AFM) to study the interactions of murine retinoschisin (Rs1) with supported anionic lipid bilayers in the presence of Ca(2+). The bilayers consisting of a single lipid, PS, and mixtures of lipids with or without PS were used. Consistent with previous X-ray diffraction studies, AFM imaging showed two distinct domains in pure PS bilayers when Ca(2+) was present. Upon Rs1 adsorption, these PS and PS-containing mixed bilayers underwent fast and extensive reorganization. Protein localization was ascertained by immunolabeling. AFM imaging showed the Rs1 antibody bound exclusively to the calcium-rich ordered phase of the bilayers pointing to the sequestration of Rs1 within those domains. This was further supported by the increased mechanical strength of these domains after Rs1 binding. Besides, changes in bilayer thickness suggested that Rs1 was partially embedded into the bilayer. These findings support a model whereby the Rs1 protein binds to PS in the retinal cell plasma membranes in a calcium-dependent manner.

Characterization of a protective Escherichia coli-expressed Plasmodium falciparum merozoite surface protein 3 indicates a non-linear, multi-domain structure.

Immunization with a recombinant yeast-expressed Plasmodium falciparum merozoite surface protein 3 (MSP3) protected Aotus nancymai monkeys against a virulent challenge infection. Unfortunately, the production process for this yeast-expressed material was not optimal for human trials. In an effort to produce a recombinant MSP3 protein in a scaleable manner, we expressed and purified near-full-length MSP3 in Escherichia coli (EcMSP3). Purified EcMSP3 formed non-globular dimers as determined by analytical size-exclusion HPLC with in-line multi-angle light scatter and quasi-elastic light scatter detection and velocity sedimentation (R(h) 7.6+/-0.2nm and 6.9nm, respectively). Evaluation by high-resolution atomic force microscopy revealed non-linear asymmetric structures, with beaded domains and flexible loops that were recognized predominantly as dimers, although monomers and larger multimers were observed. The beaded substructure corresponds to predicted structural domains, which explains the velocity sedimentation results and improves the conceptual model of the protein. Vaccination with EcMSP3 in Freunds adjuvant-induced antibodies that recognized native MSP3 in parasitized erythrocytes by an immunofluorescence assay and gave delayed time to treatment in a group of Aotus monkeys in a virulent challenge infection with the FVO strain of P. falciparum. Three of the seven monkeys vaccinated with EcMSP3 had low peak parasitemias. EcMSP3, which likely mimics the native MSP3 structure located on the merozoite surface, is a viable candidate for inclusion in a multi-component malaria vaccine.

AFM visualization of clathrin triskelia under fluid and in air

Clathrin (uniprotkb:P49951) and Clathrin (uniprotkb:P49951) bind (MI:0407) by atomic force microscopy (MI:0872)

Characterization of Nanometric Qdots by AFM and Optical Microscopy

Quantum dots (Qdots) are fluorescent nanocrystals with exceptional brightness, tunability, and photostability, and huge potential in biomedical imaging from single-particle tracking, to multicolor imaging of cell structures, to real-time in vivo imaging of whole organisms. We have undertaken a combined optical and atomic force microscopy (AFM) investigation to better understand Qdots. We have developed multimodal instrumentations around AFM platforms and total internal reflection fluorescent microscopy (TIRFM) and Raman microscopy with environmental control for simultaneous characterization of the nanoscopic structural features and optical properties of individual particles and biomedical samples. Our new instruments allow fuller observation of Qdots nanocrystal core, biocompatible coating, oligomerization states, and their optical behavior, as they interact with the environment. For example, fluorescent time traces show that the fraction of “on”-time varied from a few percent to over 80% among isolated Qdots. Our high resolution AFM topological maps and super-resolution (below the diffraction limit) TIRFM localization detection reveal a varying degree of Qdot dimerization and oligomerization on glass and mica substrates. We also demonstrate direct fluorescence spectral mapping using a newly customized and integrated Raman-AFM instrument. By understanding the relationships between their topological, mechanical, electrostatic profiles and their optical properties, Qdots can be ultimately improved toward more quantitative biomedical imaging applications.