Research in the Protein Stability, Folding, and Aggregation Laboratory is currently focused on understanding the fundamental physical mechanisms of protein stability, folding, and aggregation of disease-related and model proteins. Current research projects include studying the (1) biophysics of dystrophin and utrophin, and (2) alcohol and antimicrobial preservative-induced protein aggregation. The research group utilizes various biophysical and structural techniques that include optical spectroscopy (CD, fluorescence, and absorbance) and NMR.
Muscular dystrophy (MD) refers to a group of degenerative muscle diseases that cause progressive muscle weakness. MD affects all types of muscles. For example, decreased function of heart muscles causes heart diseases that include cardiomyopathy and congestive heart failure. Duchenne MD (DMD) and Becker MD (BMD) are two prominent types of MD, which are caused by the deficiency of a vital muscle protein known as dystrophin. These dystrophin-related diseases physically weaken patients to a state of immobility, and often cause death at an early age. Dystrophin stabilizes the sarcolemma membrane against the mechanical forces associated with muscle contraction and stretch. Mutations in dystrophin trigger the disease. Although dystrophin was identified as a key molecular player in MD 30 years ago, little is known about the biophysical mechanisms that trigger the disease at the fundamental protein level.
Utrophin, the closest homologue of dystrophin (60% sequence similarity), has been shown to compensate for the loss of functional dystrophin in animal studies, but its exact biological function is not known. It binds to actin, protects actin against its depolymerization, and interacts with dystrophin-related proteins. Utrophin is confined specifically to the sarcolemma in fetal and regenerating muscle cells. After down-regulation at birth, it is only found in the neuromuscular junctions in adult muscle cells to aid in optimal synapse transmission and to play a stabilizing role at these junctions.
In this project, we are trying to understand the biophysical and structural principles of how these two important proteins function, the effect thereon of disease-causing mutations, and whether we can develop new therapies based on the fundamental understanding of structure-function of dystrophin and utrophin.
Excipients play a major role in formulating a drug substance into a drug product. These include antimicrobial preservatives such as benzyl alcohol to prevent the accidental growth of microbes in protein formulations, aggregation suppressors such as polysorbates, reactive oxygen scavengers such as methionine, surface deadsorbents such as silicone oil, and others. In principle, excipients should be inert substances that should merely serve as the vehicle or medium for a drug or active substance, but in reality, these can interact with protein drugs causing unwanted protein destabilization and aggregation. In this project, we are trying to understand the fundamental biophysical and structural mechanisms by which excipients interact with pharmaceutical proteins using a suite of biophysical techniques that include far-UV and near-UV circular dichroism, fluorescence, isothermal titration calorimetry, differential scanning calorimetry, and 2D NMR. This work is being done as part of our Center for Pharmaceutical Biotechnology, and please contact us for any future collaborations.
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Dinen Shah: Graduate Student
Dinen’s current research is on determining the effects of lipid peroxidation products on the structure and function of a heat shock protein, 78 kDa glucose-regulated protein (GRP78), and the effect of chemical modifications such as oxidation and monoclonal antibodies.
Scott Pardee: Graduate Student
Scott's current research is on excipient interactions with pharmaceutical proteins.
Dr. Swati Bandi: Postdoctoral Fellow
Swati’s current research is on characterizing the structural dynamics of tandem calponin-homology domains of dystrophin and utrophin, and polysorbate interactions with monoclonal antibodies.
Dr. Vaibhav Upadhyay: Postdoctoral Fellow
Vaibhav’s current research is on characterizing the structure-function of dystrophin and utrophin.
Dr. Regina L. Bis: Postdoctoral Fellow
Regina’s current research is on determining the mechanisms of alcohol and antimicrobial-preservative-induced aggregation of a model protein cytochrome c and a pharmaceutical protein interferon alpha-2a.
Justine F. Molas: Graduate Student
Justine optimized the expression and purification protocols of dystrophin and utrophin, and did initial biophysical characterization of these proteins.
Darin Brown: Graduate Student
Darin worked on the expression and purification of death domains, Fas, FADD, DR4 & DR5, which are involved in apoptotic signaling pathways.
Joseph Rower: Graduate Student
Joseph worked on the effect of Hofmeister series on thermal aggregation of cytochrome c.
Dr. Surinder M. Singh: Postdoctoral Fellow
Surinder’s current research is on characterizing the biophysics of dystrophin and utrophin, two key proteins involved in muscular dystrophy.
Geoffrey Armstrong, University of Colorado Boulder
NMR experiments on utrophin and dystrophin
David Fela, NPS Pharmaceuticals
Biophysical characterization of parathyroid hormone (PTH) using NMR
Vivian Hook, University of California San Diego
Dystrophin expression in muscle cells
Thomas Lavoie, PBL Assay Science
Functional assays on interferon alpha-2a
David Nesbitt, University of Colorado Boulder
Fluorescence experiments on dystrophin and utrophin
Tara Stauffer, PBL Assay Science
Antiviral and antiproliferative assays on interferon alpha-2a
Steve Winder, University of Sheffield
Expression vectors of dystrophin and utrophin N-ABDs, and their purification and actin-binding assays
Deborah Wuttke, University of Colorado Boulder
NMR experiments on dystrophin and utrophin