The objective of this project is to study the molecular and chemical role of the amino acid, glutamine, in the process of misfolding and aggregation of proteins containing long glutamine repeats. This aberrant process, resulting in the formation of large fibrils, is a general molecular problem common to all organisms and can lead to metabolic and structural deficiencies. Recently, the process of this aggregation has come under scrutiny since the intermediates formed during fibril growth can themselves persist in the organism and interfere with normal cellular processes. Stretches of glutamine repeats fold into a b-sheet structure, which then acts as a template for aggregation by the further addition of polypeptide strands to form a long filament. Individual filaments can further associate to form multi-filament fibrils. The glutamines are thought to stabilize these structures through mechanisms involving hydrogen bonding and hydrophobic interactions, although the details of these interactions and their respective roles during the self-assembly process are still controversial. The PI has developed a peptide model system that can form a prototypical b-sheet, called a b-hairpin, that can be studied using a variety of biophysical methods to probe the role of the glutamines in fostering b-sheet, filament, and fibril formation. Three specific goals are: (1) finding conditions to stabilize intermediates in the fibril assembly pathway so that the role of glutamine interactions on growth and stability can be assessed; (2) measuring the effects of amino acid mutations on these processes to probe chemical mechanism; and (3) isotope-labeling of specific glutamines to probe directly for stable hydrogen bonding in the various intermediates. The outcome of these studies will help to understand a fundamental problem in protein misfolding and to test a variety of structural models that have been proposed for proteins containing poly-glutamine repeats.
This research has been developed as a set of projects suitable for undergraduates since the primary research efforts will be undertaken by students working to complete their thesis requirements in biology. Students engaged in this work will gain training in interdisciplinary research. This necessitates collaborative work and students will be working closely with colleagues in the Chemistry Department and collaborators at the University of Pennsylvania. Work on this project has led to the creation of lectures in protein folding and misfolding for an introductory course, more advanced lectures for an upper-level course in protein structure and function, and the development of a 7-week primary-literature-based course on protein misfolding and aggregation. The PI has a long-standing commitment to improving access to research for under-represented groups. In addition to a long-term commitment to an existing outreach program for science and writing for K-12 students from Philadelphia, he has regularly reserved space in his research lab over the summer and during the academic year for at least one student self-identified as belonging to an under-represented group. More broadly, as the director of the HHMI-sponsored programs at Haverford College, the PI is responsible for awarding student interdisciplinary research fellowships and funding internal and external research opportunities for students belonging to under-represented groups, administering several outreach programs, and supporting faculty development seminars and workshops to enhance the overall research program in the sciences.
Huntington’s Disease is a human neurodegenerative disorder that belongs to a class of diseases associated with long stretches of glutamine amino acids (polyQ). The objectives of our research were to understand how the chemical functionality of the glutamine sidechain regulates the aggregation of polyglutamine sequences, which subsequently leads to disease. Furthermore, how this aggregation takes place in the context of the flanking amino acid sequences, and in the context of the cellular milieu, are other more recent questions that we have addressed. To meet our original objectives, we developed a polyQ peptide model system that would allow us to study early assembly steps in the aggregation pathway (see Figure 1 for a cartoon depiction of the peptide folding pattern). The rationale for this approach is that it is now thought that these early assembly intermediates may be the causative agents of cell death, rather than the well-characterized plaques that had been identified in years past. A pH-triggered assembly process was initiated and the resultant aggregates were characterized using a set of biophysical approaches, including circular dichroism spectropolarimetry and Fourier transform infrared spectroscopy, a variety of sizing methods such as dynamic light scattering and analytical ultracentrifugation, and two imaging methods, including atomic force microscopy and scanning transmission electron microscopy. This work led to a publication demonstrating a detailed structural analysis of the role of the glutamines in initial interactions between individual peptide building blocks (Smith, Melanie H.,* Miles, Timothy F.,* Sheehan, Molly,* Alfieri, Katherine N.,* Kokona, Bashkim, and Fairman, Robert. 2010. Polyglutamine fibrils are formed using a simple designed beta-hairpin model. Proteins: Struct. Funct. Bioinf., 78:1971.; asterisks refer to undergraduate students). These interactions were dominated by a combination of chemical forces including the hydrophobic effect and hydrogen bonding, demonstrating a more complex interaction mechanism than had been originally postulated (involving largely hydrogen bonding alone). More recently, we became interested in how this polyQ sequence aggregates in the context of the protein implicated in Huntington’s Disease. It is known that both the N-terminal and C-terminal flanking sequences modulate the rate of aggregation, and we became particularly interested in the role of helix stability (in the N-terminal domain) and chemical composition on exacerbating or mitigating this aggregation process. We are finding that amino acids that reside at the interface of helical interactions influence this aggregation, and depend on how polar these amino acids are, with more polar amino acids inhibiting the aggregation process. Finally, in work near the end of the grant, we developed animal model systems, using fruit flies and worms, to study the aggregation using fluorescent markers, in combination with a new biophysical approach using sedimentation velocity methods. We’ve presented preliminary results at several meetings showing technical feasibility for studying such aggregation in crude extracts using sedimentation velocity, as such methodology has never been applied to complex biological systems. This work has been carried out at a small liberal arts College, in which the primary work force is composed of undergraduate students and a research assistant, who has been paid from this grant. Twelve students have worked directly on this grant, most doing this as part of their senior thesis work and as part of a ten-week summer research experience. Many other students have worked on related projects, and have benefited from exposure to the activities and findings that arose from this funded work. Students were co-authors on the published work from this grant. Students working on this project have chosen to pursue advanced degrees in biomedical work at institutions such as UC Berkeley, UC San Francisco, Univ Pennsylvania, CalTech, among others.
