Department of Biochemistry and Molecular Biology
University of Illinois, Urbana IL. Ph.D., 1987
National Institutes of Health, National Heart, Lung and Blood Institute, Bethesda, Maryland (Staff Fellow)
Publications: Click here
University of Kansas Medical Center
See video highlight from 2014 publication (scroll down) here.
Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins. Lea WA, O'Neil PT, Machen AJ, Naik S, Chaudhri T, McGinn-Straub W, Tischer A, Auton MT, Burns JR, Baldwin MR, Khar KR, Karanicolas J, Fisher MT. Biochemistry. 2016 Sep 6;55(35):4885-908. doi: 10.1021/acs.biochem.6b00293. Epub 2016 Aug 19.
Protein folding on biosensor tips: folding of maltodextrin glucosidase monitored by its interactions with GroEL. Pastor A, Singh AK, Fisher MT, Chaudhuri TK. FEBS J. 2016 Aug;283(16):3103-14. doi: 10.1111/febs.13796. Epub 2016 Aug 1.
Following Natures Lead: On the Construction of Membrane-Inserted Toxins in Lipid Bilayer Nanodiscs. Akkaladevi N, Mukherjee S, Katayama H, Janowiak B, Patel D, Gogol EP, Pentelute BL, John Collier R, Fisher MT. J Membr Biol. 2015 Jan 13
Probing structurally altered and aggregated states of terapeutically relevant proteins using GroEL coupled t bio-layer interferometry. Naik S, Kumru OS, Cullom M, Telikepalli SN, Lindboe E, Roop TL, Joshi SB, Admin D, Bao P, Middaugh CR, Volkin DB, Fisher MT. Protein Sci. 2014 Oct:23(10):1461-78
Major Research Interests
Projects: Structural biophysics of transient and captured protein states (Anthrax Toxin Pore translocation complex), Protein Folding and Pharmaceutical Drug Development.
Introduction to our research:
Our understanding of protein folding inside the cell has advanced significantly over the last two decades. Biochemists are now aware that cellular protein folding is, in most cases, assisted by other essential proteins called molecular chaperones. With this new knowledge, we are beginning to appreciate the critical role the protein homeostasis plays in cell viability and Human disease. From a medical standpoint, understanding cellular folding is extremely important because valid estimates gleaned from molecular genetic databases indicate that between 30 TO 50% OF THE TOTAL NUMBER OF HUMAN DISEASES AT ONE TIME ARE CAUSED BY SOME SORT OF PROTEIN FOLDING DEFECTS. Among the most well-known folding diseases are Alzheimer's disease, Parkinson's disease, Huntington's, ALS, and Cystic Fibrosis. In addition these more recognizable diseases, we are now also aware that bacterial/viral protein toxins have to undergo folding changes that result in disease. In these cases, numerous bacterial/viral protein toxins (Anthrax, Diphtheria, Botulinum, Ricin, Shiga toxin, influenza, MRSA) literally change folding shapes during infection.
To make any headway on these broad disease fronts, our research efforts must advance from the preliminary discovery phase obtained from genomics or proteomics efforts toward the application to devise targeted therapies to tackle each of the disease states one protein at a time. In order to develop small molecule stabilizers for proteins, we use the high affinity form of the chaperonin protein to detect partially folded proteins (dynamic or metastable). We use this interaction to construct a moderate throughput chaperonin based platform technology using label free technologies Surface Plasmon Resonance (SPR) and Biolayer interferometry (BLI) to screen and validate chemical compound libraries. The latter label free method is the primary platform that we routinely use in the laboratory. The research in the Fisher lab is focused on taking the vast knowledge describing molecular chaperone function and applying this data to establish broad based research tools and approaches to eventually aid in the identification and design of the next generation of small molecule protein drugs to ameliorate Protein Folding Diseases. We have also designed novel protein toxin based platforms to recapitulate and detect endosomal like unfolding/refolding transitions of occur as bacterial or viral toxin gain entry through cellular endosomal membranes.
Major research projects:
The Spear of Anthrax Comes into View. EM structure of the Anthrax Toxin Pore translocon.
The Fisher laboratory along with Ed Gogol from University of Missouri Kansas City and R. John Collier and colleagues from Harvard have constructed the first structural images of the anthrax pore translocon "spear" inserted into a nanodisc lipid bilayer. This pore initially binds cell surface receptors as benign lethal toxin-prepore complexes. The cell then engulfs this trojan horse, transforming the prepore into the pore, allowing the passenger toxins (lethal factor and edema factor) to unfold and thread through the pore interior to disrupt cellular function. The trick to obtain this structure followed Nature's lead which entailed tethering the prepore-lethal toxin complex, followed by transformation to the pore before assembling the nanodisc around the hydrophobic pore tip. The EM data was collected at the NRAMM (National Resource for Automated Molecular Microscopy) Scripps facility (Akkaladevi Protein Science) with Brigit Carragher and at the NCMI (National Center for Macromolecular Imaging) Baylor facility (S. Ludtke and W. Chiu).
Schematic procedure for constructing purified Anthrax translocon pores inserted into lipid nanodiscs.
Akkaladevi et al., (2013) Protein Sci. 22:492-501.
Our structure work with the Protective antigen pore translocon has yielded the first low resolution structures of this membrane protein inserted into lipid nanodiscs using single particle analysis with images from negative stain EM and cryoEM. The figure below shows the progressive improvements for obtaining the structure of the anthrax toxin pore translocon (Protective antigen) using Electron microscopy single particle analysis.
Even these low resolution EM structures reveal some important structural features that lead to some functional insight. For example, the positioning of the phe clamp loop (discovered by Krantz and Collier) appears to be positioned to constrain the opening of the lumen at pH 7.0. In addition, the interior lumen (and exterior) of the anthrax pore translocon does not appear to be a smooth barrel and displays various constrictions, bulges and vestibules. By taking account of its complex electrostatic interior, our next goal use molecular dynamic approaches (MMFF) to model the interior lumen and exterior barrel surface (continuing collaboration with Srayanta Mukherjee, Wonpil Im).
Recapitulating Endosomal Transitions on Label free surfaces :
We have also used the label free surface Plasmon resonance (SPR) and biolayer interferometry (BLI) platforms to measure real time unfolding-refolding transitions of this pore translocon. We constructed the complete anthrax toxin complex using the same methodology we used to construct and purify PA pores inserted into nanodiscs. With the label free surfaces, the SPR or BLI signals can detect the real time transitions of the prepore to pore transitions (see figure below for BLI). When the partial anthrax toxin is constructed on the label free surfaces (e.g. attached LFN-PA prepore), the acidification of the solution mimicks the late endosomal pH (5.0) and results in the transition of the prepore to pore conformation.
The figure to the right illustrates the observed signal response that arises from the PA prepore to pore transition alone. The use of biolayer interferometry biosensor tips allowed us to remove the transitioned Pore translocons from the surface and transfer these to Electron microscopy grids (bottom of the figure) to view the transitioned pore. The presence of a lipid micelle shows that the pores no longer aggregate and can easily be resolved as soluble individual pores.
Designing Chaperonin-Based HTS platforms to identify Protein folding disease stabilizers
Identifying pharmacological chaperones: A number of protein folding diseases develop because missense mutations shift the equilibrium distribution between native and partially denatured species. In addition to missense mutation induced changes, equilibrium shifts toward misfolded protein populations also can result from changes in the solution environment or through the simple overexpression of the native protein. An emerging strategy and potential therapy for ameliorating some of these misfolding diseases is to employ so called "pharmacological chaperones" or more appropriately, protein stabilizers. These stabilizers or chemical chaperones bind and stabilize native states in equilibrium with disease causing states and prevent, through mass action effects, the accumulation of the deleterious misfolded protein. We are now developing label-free systems to rapidly identify and test for plausible pharmacological chaperones. As illustrated below, the chaperonin will kinetically bind and partition partially folded states of proteins. If a stabilizer is present, this partitioning is decreased or is no longer observed. This is the basis of our generic screening procedure.
"Using Natures sentries to capture the seeds of destruction:".
Naik, et al., "On the Design of Broad Based Screening Assays to Identify Potential Pharmacological Chaperones of Protein Misfolding Diseases." Current Opinions in Medicinal Chemistry vol 12 (22): 2504-22.
User Presentations for fortébio (a division of Pall Life Sciences)
M Fisher presentation at ~ 20 min into Webinar.
Mark T. Fisher, Ph.D.