Kristin N. Parent, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The amino acid sequence of a protein determines its fold. However, small changes such as single amino acid substitutions or deletions can cause misfolding, and subsequent aggregation. Protein misfolding directly causes several devastating human diseases. Molecular chaperones assist the folding of many proteins, especially those prone to misfolding and aggregation. However, how chaperones distinguish between incorrectly and productively folding proteins is unclear. The mechanism of substrate specificity of the major bacterial chaperone complex, GroEL/ES, will be investigated. The major aim of this proposal is to select and characterize mutants of GroEL/ES that more efficiently fold substrate proteins. The coat protein of phage P22 will be the model substrate used to isolate the chaperone mutants because several single amino acid substitutions convert coat protein to become a substrate for GroEL/ES, though with limited efficiency. GroEL/ES mutants will be selected that can fold the coat protein mutants at high temperatures. The ability of the GroEL/ES mutants to fold a variety of substrate proteins will be determined both in vivo and in vitro to gain a generalized understanding of how these chaperone proteins recognize substrate polypeptides. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Viral infection significantly impacts all forms of life. Since every virus and bacteriophage requires correct organization of its capsid proteins (CPs), the mechanisms by which these CPs assemble must ultimately be determined in order to devise efficacious methods to circumvent infection or its spread. Bacteriophage P22 shares features of assembly and a high degree of structural conservation among the major CPs of numerous dsDNA viruses, including the human herpesviruses, and will serve as a model system in the proposed research. We will study the stages of infection in P22-infected Salmonella, employing both electron tomography of samples treated by High Pressure Freezing and Freeze Substitution (HPF/FS) and fluorescence microscopy. Initially, our focus will be on visualizing early events of infection, including P22 attachment to cells and subsequent release of the minor proteins gp16, gp7, and gp20 into cells. Ultimately, these methods will help us characterize many stages of the virus life cycle, including those that occur during the end of the cycle such as procapsid assembly and maturation. In addition, we propose to employ electron cryo-microscopy on single particles to determine the structural transitions that occur in the major capsid protein as the virion matures. Adding and mastering these powerful techniques to my repertoire will bolster my path towards a faculty position at a top tier institution.

? DESCRIPTION (provided by applicant): Viruses infect their respective hosts efficiently through a regulated process of recognizing specific receptors and subsequently transferring genomic material across cell membrane barriers. To understand the underlying mechanisms that control virus infection, it is important to characterize viral events in a biologically relevat manner. I have been able to study Sf6 infection in the context of its host, Shigella flexneri, through classic phage analyses and have identified two receptors important for Sf6 infection (S. flexneri outer membrane proteins A & C, Omps A & C). OmpA is the preferred receptor but Sf6 can utilize OmpC as an alternate route for infection. I plan to study the interactions between Sf6 and S. flexneri to determine regions in both the phage proteins and the Omps that are critical for proper infection. I also plan to extend our understanding of Sf6 infection dynamics since this phage mimics infection in vitro by binding and delivering its genome into host-derived Outer Membrane Vesicles (OMVs). This provides an ideal system for using cryo-electron tomography to visualize intermediates that arise during Sf6 infection, and variant receptors as the OMVs are considerably thinner (~100 nm thick) compared to whole Shigella cells (~2000 nm thick), which are currently outside the limits of effective cryo-tomography. In vitro genome ejections are also possible using purified receptors. This makes Sf6 one of a few model systems to correlate structural transitions that arise during host cell recognition and resulting genomic transfer, and s currently the only model system for Adenovirus with alternate receptors known. We developed a time-lapse fluorescence assay that monitors real time genome ejection in vivo at the single particle level. This, combined with decades of groundwork on bacteriophage genetic manipulation and biochemical/biophysical characterization, allows us to study the process of Sf6 infection from several different angles and will provide insight into the generalized mechanisms by which all viruses recognize, and infect hosts. Sf6, as well as many other phages and eukaryotic viruses, has several so-called ejection proteins that are passed from the virion into the host during infection. Ejection proteins of Sf6 are of particular interest since they interact intimately with the host, which is a human pathogen. The location of ejection proteins pre- or post-infection has yet to be determined for any phage, but it is likely that they play a role in protecting the genome as it is injected into the host. Therefore, we predict the ejection proteins to be released in a sequential manner, and to affect the rate and efficiency of genomic transfer. We will test this hypothesis through biophysical approaches.

PROJECT SUMMARY Recently, single particle cryo-electron microscopy (cryo-EM) combined with 3-D reconstruction has emerged as a revolutionary tool for solving high-resolution 3-D structures of viruses and macromolecular complexes. The rapidly increasing number of near-atomic resolution (3-4?) structures solved using cryo-EM has allowed unprecedented atomic level understanding of fundamental cellular processes and viral infections. To obtain near-atomic resolution cryo-EM structures, requires the collection of high-resolution image data using state-of- the-art imaging resources, including both a high-end transmission electron microscope and a direct electron detector. The direct electron detectors not only improve image resolution and contrast, but also record movies for subsequent computational correction of electron beam-induced, sample movements during exposure. The increased image contrast and resolution are essential for solving near-atomic resolution structures of small protein complexes. However, the high cost to purchase and maintain a state-of-the-art cryo-EM resource, with both a high-end electron microscope and a direct electron detector, precludes many cryo-EM investigators from having access to these new techniques. Here, the creation of a Midwest Consortium for High- Resolution Cryo-electron Microscopy is proposed to provide access to such high-resolution data collection capability for cryo-EM laboratories without access to such resources. This consortium will consist of 4 investigators from the host institution (Purdue Univ.) which will maintain the high-resolution data collection resource (Titan Krios 300kV FEG microscope with phase plate, energy filter, and direct electron detector) and will provide services to 11 investigators from 10 partnering institutions (Boston Univ. School of Medicine, Michigan State Univ., Penn State College of Medicine, Rutgers Univ., Univ. of Alabama at Birmingham, Univ. of Colorado Boulder, Univ. of Kansas, Univ. of Missouri, Univ. of Vermont, and Virginia Commonwealth Univ.). The collective experience of the Purdue facility staff, faculty and onsite service engineer, in high-resolution cryo-EM imaging, will ensure that the facility operates at peak performance with minimal service interruptions. The high-resolution data collection capabilities established at Purdue and accessible to the partnering labs, will allow these investigators, who are all, except for the 3 new investigators, NIH-funded, to overcome the resolution barrier and facilitate discovery within their own cryo-EM projects on a range of structures, such as, bacterial pathogen adhesion proteins, human viruses, Huntington's Disease proteins, synaptic vesicle proteins, chemoreceptor signaling complex, etc. The Consortium will provide comprehensive support to the cryo-EM projects, including shipping samples, preparing sample grids, collecting high-resolution single particle and tomography images, processing raw movies, and transferring data back to the partners' labs. The data collection services will range from full data collection where partners simply mail in the samples and then receive the image data, to hands-on operations of the cryo-EM by partners trained by the host facility's staff.