The Imaging Sciences Laboratory is involved in a major collaborative research effort with the NIH Institutes involving the use of image processing techniques and advanced computational techniques in structural biology to analyze electron micrographs NMR spectra and other data with the goal of determining macromolecular structures and dynamics. Recent efforts have concentrated on the 3D reconstruction, analysis and interpretation of the structures of icosahedral virus capsids in addition to structure determination and analysis of isolated and complexed proteins and nucleic acids. Ongoing research involves analyses of structures related to papillomavirus and RNA containing virus capsids. In addition, we have also been developing computational tools for the study of the structure and dynamics of biological macromolecules using Nuclear Magnetic Resonance (NMR) and other data. We develop and maintain the Xplor-NIH software package for structure determination, which is used in the NMR labs in the Institutes, and also worldwide. Human papillomavirus (HPV) has been implicated as the causative agent in cervical and other epithelial cancers. The major capsid protein (L1), initially forms a loosely connected procapsid which, under in vitro conditions, condenses over several hours into the more familiar 60 nm-diameter papillomavirus capsid. Current work involves collaborations with a goal to engineer a better HPV vaccine. HPV also contains a minor capsid protein (L2). The L1 sequence is not highly conserved across all types of HPV. L2, on the other hand, is highly conserved. Current (L1 containing) vaccines are roughly 85% effective because they contain a mixture of HPV subtypes. However, since L2 is highly conserved, a vaccine based on L2 could provide close to 100% efficacy. Our goal is to study the structure of a better vaccine candidates (i.e. to try to achieve closer to 100% effectiveness). This is an example of Hi-Risk, Hi-Reward research. We have been looking at a chimera papillomavirus, which contains L2 inserted into L1 to produce pseudocapsids. To date, only low-resolution 3D reconstructions have been achieved due to heterogeneity in the sample. Hopefully, future samples will permit high resolution analysis. Another long-term structural project studies double-stranded RNA containing viruses. Infectious bursal disease virus (IBDV), a non-enveloped, double-stranded (ds)RNA virus with a T=13 icosahedral capsid. In IBDV-infected cells, the assembly pathway results mainly in mature virions that package four dsRNA segments. We used cryo-electron microscopy, cryo-electron tomography and atomic force microscopy to characterize these IBDV populations. The VP3 protein was found to act as a scaffold protein by building an irregular, 40 -thick internal shell without icosahedral symmetry, which facilitates formation of a precursor particle, the procapsid. Analysis of IBDV procapsid mechanical properties indicated a VP3 layer beneath the icosahedral shell, which increased effective capsid thickness. Whereas scaffolding proteins are discharged in tailed dsDNA viruses, VP3 is a multifunctional protein. In mature virions, VP3 is bound to the dsRNA genome, which is organized as ribonucleoprotein complexes. IBDV is an amalgam of dsRNA viral ancestors and traits from dsDNA and single-stranded (ss)RNA viruses. In collaboration with researchers in NIDDK we calculated the structure of a dimer of Huntingtin peptides associated with a membrane surface. It is hypothesized that Huntingtin fibril formation is initiated when two monomers attach to a membrane surface and diffuse on this surface to find each other. The poly-glutamine regions of Huntingtin which lie adjacent to the lipid-attractive alpha helices at the N-terminal tails then interact, detach from the lipid and find other Huntingtin subunits to aggregate and eventually become the pathogenic fibrils associated with Huntington's disease. The dimer structure structure was determined using distances derived from electron paramagnetic resonance spectroscopy (EPR) data. However, importantly, the effect of the membrane was included using an implicit solvent energy term developed previously. In this context, it was found that the EPR data were consistent with a membrane-associated dimer where the membrane-subunit interactions dominate those between the subunits. Such a structure allows for the poly-glutamine tails to be in close proximity while the alpha helices are still associated with the membrane. Solvent paramagnetic relaxation enhancement (sPRE) data serve as an NMR probe of distance to solvent accessible surface. This observable is represented as in integral of 1/r6 over entire volume occupied by the paramagnetic cosolute, where r is the distance between a volume element and the nucleus probed by the experiment. This integral cannot directly by efficiently computed. However, it can be transformed into an integral over the cosolute-excluded molecular surface using the divergence theorem. This molecular surface can be efficiently tessellated into connected triangles, which can be summed over to calculate sPRE values when given a molecular structure. This expression for sPRE can be approximately differentiated such that sPRE data can be used in gradient-based structure determination algorithms. We have shown that this data can be very useful in calculating the structures both of rigid proteins, and those which are sampling multiple substates.
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