Structural Biology and Biophysics Research
Modern biological sciences are enriched by the revolution in structural biology. Advances in techniques such as electron microscopy (EM), X-ray crystallography and NMR, coupled with modern biophysical approaches make possible an unprecedented ability to understand fundamental biological problems at the molecular level. The UAB Department of Microbiology has enviable strengths in all of these disciplines. Our faculty apply these techniques to a range of biological problems, including viral entry, replication and assembly, antiviral drug design, autoimmune disease and cancer. In addition to the remarkable strength of our structural biology research, our faculty offer advanced courses centered on the basics and applications of structural techniques in cell and molecular biology.
Terje Dokland, Ph.D.
Structure and assembly of viruses and bacteriophages
My lab uses a hybrid approach combining cryo-electron microscopy (cryo-EM), three-dimensional reconstruction, X-ray crystallography, NMR and other biochemical and biophysical approaches to study the structure, assembly and function of viruses, bacteriophages and proteins involved in bacterial pathogenicity. Cryo-EM allows the imaging of biological material in its native stain, in the absence of fixation, staining and drying artifacts and can be combined with 3D reconstruction methods, such as electron tomography. Our main project is focused on the role of bacteriophages in the mobilization of pathogenicity islands (SaPIs) in Staphylococcus aureus and the mobilization and assembly of P2-like bacteriophages found in many environmental and clinical E. coli strains, including E. coli O157:H7.
Todd Green, Ph.D.
Dr. Green’s lab uses structural techniques to study proteins from negative-stranded RNA viruses that are involved in polynucleotide synthesis. In order to gain a better understanding of the replication cycle of this class of viruses, my lab is studying the molecular structure of vesicular stomatitis virus (VSV), a prototype of this group. Using state-of-the-art techniques, such as x-ray crystallography and cryo-electron microscopy, we have observed atomic-level snapshots of how the viral capsid protein encapsidates and protects the genomic RNA, how this template is accessed by the viral polymerase co-factors, and how the capsid structure is affected by specific RNA sequences. Moving forward, we continue to investigate the mechanisms of viral transcription and replication by structural and biochemical techniques. Better understanding of these processes will yield new avenues for future therapeutic design against this group of viruses.
Michael Niederweis, Ph.D.
Nanopore DNA sequencing with MspA
Our laboratory is working on the development of new, fast, and inexpensive DNA sequencing technology. We discovered a porin from Mycobacterium smegmatis (Niederweis et al., 1999) whose short and narrow constriction zone (Faller et al., 2004) and extreme robustness to temperature and harsh chemicals (Heinz et al., 2003) turns out to be ideal for nanopore sequencing of DNA (Manrao et al., 2012, Derrington et al., 2010, Butler et al., 2008). In this method, single-stranded DNA is driven through the MspA pore by an electrical field. Each nucleotide modulates the ion current in a sequence-specific manner when DNA moves through the pore. The focus of our laboratory is to fine-tune the MspA pore for DNA sequencing by making a single chain MspA pore (Pavlenok et al., 2012) and introducing asymmetrical mutations to improve the capture rate of DNA, reduce the translocation speed and enhance base recognition.
- Butler, T. Z., M. Pavlenok, I. M. Derrington, M. Niederweis & J. H. Gundlach, (2008) Single-molecule DNA detection with an engineered MspA protein nanopore. Proc Natl Acad Sci U S A 105: 20647-20652.
- Derrington, I. M., T. Z. Butler, M. D. Collins, E. Manrao, M. Pavlenok, M. Niederweis & J. H. Gundlach, (2010) Nanopore DNA sequencing with MspA. Proc Natl Acad Sci U S A 107: 16060-16065.
- Faller, M., M. Niederweis & G. E. Schulz, (2004) The structure of a mycobacterial outer-membrane channel. Science 303: 1189-1192.
- Heinz, C., H. Engelhardt & M. Niederweis, (2003) The core of the tetrameric mycobacterial porin MspA is an extremely stable beta-sheet domain. J. Biol. Chem. 278: 8678-8685.
- Manrao, E. A., I. M. Derrington, A. H. Laszlo, K. W. Langford, M. K. Hopper, N. Gillgren, M. Pavlenok, M. Niederweis & J. H. Gundlach, (2012) Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat Biotechnol 30: 349-353.
- Niederweis, M., S. Ehrt, C. Heinz, U. Klöcker, S. Karosi, K. M. Swiderek, L. W. Riley & R. Benz, (1999) Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis. Mol. Microbiol. 33: 933-945.
- Pavlenok, M., I. M. Derrington, J. H. Gundlach & M. Niederweis, (2012) MspA nanopores from subunit dimers. PLoS One 7: e38726.
Jan Novak, Ph.D.
Dr. Novak’s research interests in Structural biology and biophysics include the structure-function studies of anti-glycan antibodies in autoimmune diseases and cancer, enzymes and regulation of O-glycosylation pathways, and the impact of variants and isoforms of specific glycosyltransferases on enzyme activities and resultant glycosylation.
Peter Prevelige, Ph.D.
Virus Structure and Assembly
My laboratory uses a variety of biophysical and structural approaches to approach fundament questions in virus structure and assembly. We are particularly interested in questions of form determination, for how icosahedral viruses assemble from subunits which high fidelity, or why some viral capsid such as HIV adopt different morphologies. Among the techniques that we utilize are: cryo-EM, NMR, mass spectrometry, single molecule spectroscopy, light scattering, and kinetic analysis.
Virus Based Bio-Nanotechnology
Virus particles can be viewed as nanoscale structural scaffolds precisely defined down to the atomic level. Well understood techniques of protein chemistry and molecular biology provide facile tools for the selective modification of individual amino acids. These modified viral particles can be used to display targeting ligands for biomedical imaging and delivery (theranostics) applications or alternatively as a route to well-defined nanoscale materials. Our virus based bio-nano projects utilize our detailed knowledge of the structure and assembly of the 60nm capsid of the bacteriophage P22 to address both of these classes of applications.
Jamil S. Saad, Ph.D.
We employ a set of structural biology, biophysical and biochemical tools to understand how HIV proteins interact with cellular proteins and membranes during the virus replication cycle. These methods allow us to identify key protein-protein and protein-lipid interactions. Among the diverse techniques we utilize are: Nuclear magnetic resonance (NMR), X-ray crystallography, isothermal titration calirometry (ITC), surface plasmon resonance (SPR), analytical ultracentrifugation (AUC), florescence, mass spectrometry (MS), and computational biology. State-of-the-art high-resolution NMR instruments (850, 700, 600 and 500 MHz NMR) equipped with cryogenic probes are utilized. My lab has developed new NMR strategies to study protein-protein and protein-lipid interactions. Additionally, we developed a rapid NMR-based assay that, in a single experiment (20 minutes), enables us to: (i) detect direct binding, (ii) determine binding affinity, (iii) identify the binding site on the protein, (iv) determine the stoichiometry of binding, and (v) assess whether small molecules block interactions.
My research involves using X-ray crystallographic techniques to study viral protein structures, and taking this structural biology approach to develop potential inhibitors of viral proteins that are implicated in viral assembly and infection. We currently work on influenza virus, negative strand RNA viruses, flaviviruses and hepatitis viruses.
Mark R. Walter, Ph.D.
Crystal structure analysis and biophysics of cytokine-mediated receptor assembly: A major focus of the lab is elucidating crystal structures of cytokines bound to their cell surface receptors. These studies provide the framework for detailed biochemical and cellular characterization of how cytokines assemble a “signaling competent” complex leading to cellular activation. The goal of these studies is to extend our understanding of basic signaling mechanisms. These findings are then applied to complex problems in human disease. As described in the immunology section of the website, we are currently using what we have learned to understand interferon signaling in lupus, and design novel vaccine strategies to prevent human cytomegalovirus (HCMV) infection, which is responsible for serious birth defects in children whose mothers are infected while pregnant. See below for recently determined crystal structures:
- Logsdon et al. Structural basis for receptor sharing and activation by interleukin-20 receptor-2 (IL-20R2) binding cytokines. P.N.A.S. 109 (31) 12704-12709 (2012).
Structural biology of viral proteins that disrupt cytokine signaling: An ongoing goal of the laboratory is to design molecules that enhance or prevent cytokine signaling. One way to understand how to manipulate cytokine signaling is to understand the mechanisms used by viruses to accomplish this task. Pox and herpes viruses have designed novel proteins to disrupt cytokine signaling when they infect cells. Structure-function studies on these molecules provide considerable insight into the mechanisms used by virus to evade the host immune system. Furthermore, these strategies might also be used to control dysregulated cytokine signaling observed in autoimmune diseases. See
- Yoon et al. Epstein-Barr Virus IL-10 Engages IL-10R1 by a Two-step Mechanism Leading to Altered Signaling Properties. J. Biol. Chem. 26586-26595, 2012 and
- Nuara et al. Structure and mechanism of IFN-γ antagonism by an orthopoxvirus IFN-γ Binding Protein P.N.A.S. 105, 1861-1866, (2008).