As a growing number of proteins are identified as intrinsically disordered--consisting entirely of or containing long stretches of regions that do not populate well-defined secondary or tertiary structures--there is a corresponding growth in interest in these proteins. They challenge the central dogma in modern structural biology that function follows from structure. Gaining an understanding of how biological function is achieved is challenging in disordered proteins, in part due to the fact that they are inherently heterogeneous with multiple conformations and species populated simultaneously and with different lifetimes. In contrast to traditional ensemble methods, the single molecule biophysical approaches used in this research are uniquely suited to characterize heterogeneous molecular species in a wide range of solution conditions and associated with relevant binding partners. This project focuses on the protein tau, a microtubule binding protein that is entirely disordered in solution and only partly structured when functionally bound to microtubules. In a systematic study using single molecule experimental and computational approaches, functional fragments of tau--such as the microtubule-binding domain--will be characterized both independently of and in the context of the entire protein chain. This will allow for a precise determination how the conformations and dynamics of specific regions are coordinated, as engineered perturbations to one region of the protein will be assayed for their effects on the functionality of distant regions. Furthermore, the studies described here may serve as a model for designing studies of other intrinsically disordered proteins, and help elucidate how transient structure formation and long range interactions generate function in this intriguing group of proteins.

There are a number of educational and outreach activities that are integral to the PI's research goals. The PI actively works to involve undergraduate students in research. The research projects are ideal for undergraduate research; several undergraduate students are already actively involved, including one from a summer program for underrepresented minorities, and another who will spend the summer and complete her senior thesis in the PI's lab in the upcoming year. Another component is the PI's effort in the recruitment of underrepresented students to graduate study in the sciences at Yale. In addition to serving on the departmental diversity committee, the PI has attended and spoken at conferences aimed at these minority students, including the Annual Biomedical Research Conference for Minority Students (ABRCMS) and the Conference for Undergraduate Women in Physics at Yale. Lastly is a multifaceted plan to improve interdisciplinary training opportunities for students interested in biophysical science. This includes the development and expansion of a course designed to teach mathematical methods to biology undergraduate and graduate students. The course covers analytical and numerical methods needed for analyzing biological data, with a strong emphasis on learning to use MATLAB software for solving and modeling complex biological problems. Synergistic with this course development, the PI is involved with a new training program in Biological Physics that recruits talented graduate students with an interest in interdisciplinary research, but lacking the academic training in biology or physics.

Project Report

Intellectual Merit The overall goal of this research was to gain insight into the basic structural and functional features of the protein tau. Tau’s primary function is to stabilize microtubules (MTs); its aggregation into insoluble, fibrillar deposits is implicated in neurodegeneration. Tau lacks stable secondary and tertiary structure, which makes its structural characterization extremely challenging. Our approach was to use single molecule Förster resonance energy transfer (smFRET), a fluorescence method which allows for coarse-grain conformational characterization even in the absence of stable structure. We had three major objectives in our research and our primary findings for each are outlined below; Objective 1: Determine tau structures in solution and in neurofibrillary tangles smFRET was used to characterize the structure of tau in the absence and presence heparin, a molecule which induces aggregation. Tau was labeled at twelve different sets of residues in order to probe specific domains and the resulting smFRET measurements enabled a detailed structural model of the conformational changes that occur upon heparin binding. A particularly important result was the loss of long range contacts between the two termini and between each terminus and the MT-binding domain, accompanied by a compaction of this domain (Figure 1). We developed coarse-grained computational methods which incorporate experimentally derived smFRET parameters as distance constraints. Such methods are particularly important to obtain structural information for large, disordered proteins where all-atom simulations are currently infeasible. Objective 2: Measure the conformations of MT bound tau Our goals evolved to focus primarily on the interaction between tau and tubulin, driven by our finding that tau binds with high affinity to tubulin dimers and that the tau-tubulin has been largely unexplored. Using tau point mutants, we made the novel observation that the point variants impact interactions between tau and tubulin much more strongly than those between tau and MTs (Figure 2). Importantly, these mutants are also deficient in their ability to polymerize tubulin, leading us to propose a novel mechanism for tau-mediated MT polymerization which focuses on the reversible interaction between tau and tubulin. Using smFRET as in Objective 1, we have a preliminary map of the topology of tau bound to tubulin which will be used to generate the most detailed picture of the interaction of tau with its native binding partner to date. Objective 3: Determine the role of surface interactions in tau aggregation Using a novel assay, we observed that anionic vesicles are extremely efficient as inducers of tau aggregation. Using the same mutants described in Objective 2, we explored the link between increased aggregation propensity and tubulin-polymerization capacity and found that contrary to dogma in the field, there was not a straightforward relationship between these two parameters. A better understanding of this relationship is the focus of ongoing research. Broader Impacts Training: The educational aspects of this proposal are closely tied to the research objectives in that each student involved in this project receives interdisciplinary training in biology, physics, and chemistry. The skills acquired include: protein expression and purification; generation of mutants; fluorescence labeling and characterization; single molecule measurements; data analysis and interpretation; optical alignment and instrument development; writing manuscripts and oral presentation of research. A total of 5 graduate students (1 URM; 2 female) and 7 undergraduates (2 URM; 5 female) from the Molecular Biophysics and Biochemistry, Chemistry, and Physics Departments at Yale have been supported by this grant for various periods of their training. Both graduate and undergraduate students had opportunities to present their research (and thus assisted me in dissemination efforts) in a variety of platforms ranging from local journal clubs to national scientific meetings. In addition, all of the graduate students had the opportunity to oversee/mentor other lab members (first year graduate students during their research rotations or undergraduate lab members). Outreach: Outreach to a broader scientific community has taken the form of dissemination of results during visits to Universities and at scientific meetings (I gave >25 talks relevant to this research). Specific outreach to undergraduates comes from research talks at undergraduate institutions and to undergraduate science societies at Yale (undergraduate biology/physics students; panel about "Graduate School Admission"). Moreover, I was actively involved in efforts to improve the recruitment and retainment of women faculty in the sciences. Three of the five students who have obtained PhD’s in my lab are women: one is an assistant professor of Chemistry at SUNY-Purchase and the other two are post-doctoral associates at Princeton and Yale. I continue to serve as a source of support and advice for them as the progress in their careers. In addition, I participated in panel/outreach sessions aimed at young women in the sciences, include the Yale Conference for Undergraduate Women in Physics, the Yale-Pfizer Mentoring Program through Women in Science at Yale, and "Female Scientists in Academia" held at the University of Pennsylvania.

National Science Foundation (NSF)
Division of Molecular and Cellular Biosciences (MCB)
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Kamal Shukla
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Yale University
New Haven
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