Gary W. Daughdrill - US grants

CORE ABSTRACT NOT AVAILABLE

protein protein interaction; structural biology; protein structure function; protein folding; biochemical evolution; natural selections;

functional /structural genomics

functional /structural genomics

The objective of this project to is study structural variations in a family of intrinsically unstructured proteins (IUP). Based on the PI's previous work, and the work of others, it is now clear that IUPs form a diverse set of protein families that can elicit biological function using a variety of mechanisms. This project has two aims: (I) Determine structural ensembles for a set of homologous proteins that are intrinsically unstructured. In this specific aim, paramagnetic relaxation enhancement will be used to estimate long-range interatomic distances that can be used to restrain structure calculations. The structural ensembles will then be validated using residual dipolar couplings and small angle x-ray scattering. (II) Determine the relationship between sequence identity and structural similarity for a set of homologous proteins that are intrinsically unstructured. In this specific aim, eigenvector decomposition of protein contact maps will be used to identify similarities between the structural ensembles. This data will then be correlated with genetic distance matrices to determine the percent sequence identity necessary for structural similarity. Experiments in this specific aim will also test the importance of amino acid composition for specifying the structural ensemble. Successful completion of both specific aims will facilitate the development of models of protein evolution for intrinsically unstructured proteins that can be used to estimate their dynamic structures based on sequence identity.

The PI has a strong record of advancing discovery and understanding while promoting teaching, training, and learning. This is evidenced by the three peer reviewed publications that have undergraduate students as authors and five peer reviewed publications that have authors from underrepresented groups. The PI is also very committed in disseminating scientific information to general public. In the summer of 2004, the PI participated in a state funded workshop for secondary education to science teachers. The PI gave lectures on the general principles of protein structure and function as well as a tutorial for using molecular graphics programs in the classroom. This project will involve two undergraduate students and a female research technician and the PI will continue his efforts to involve science teachers in basic scientific research by holding an annual workshop whose purpose would be to introduce science teachers to the latest breakthroughs and make them aware of new web-based tools for scientific discovery.

DESCRIPTION (provided by applicant): An important outcome of the recent protein structure initiative is the discovery of numerous protein families that do not form compact rigid structures. These intrinsically unstructured proteins (IUPs) are common in nature and disrupting their function can also result in the onset of certain diseases. We believe that to understand the function of IUPs it is crucial to generate realistic structural ensembles. Such ensembles are difficult to generate for IUPs where experiments predict broad, heterogeneous ensembles of structures that undergo large-scale conformational fluctuations. Experimentally restrained ensembles of loop regions in structured proteins are also difficult to reliably compare to the equilibrium ensemble. We are interested in testing two hypotheses through the use of combined computational and experimental approaches: (i) Experimental data for IUPs which are based on average measurements can be used to generate useful structural ensembles. (ii) These ensembles must be properly weighted in the equilibrium distribution to be useful for understanding protein function. In an effort to expand our understanding of how well experimentally restrained ensembles of unstructured proteins represent the equilibrium ensemble, coarse-graining will be used to generate large ensembles that are restrained using average distance and dihedral angle measurements from nuclear magnetic resonance (NMR) spectroscopy experiments. These structurally diverse ensembles will extend previous work in this area by more thoroughly sampling conformational space. These ensembles will then be re-weighted using non-rigorous methods based on fitting data from small angle x-ray scattering and residual dipolar couplings. We will also extend a rigorous re-weighting approach to loops in structured proteins. These goals will be accomplished through the following Specific Aims: Aim 1: Generate large NMR ensembles for IUPs using coarse-graining. Aim 2: Re-weight NMR ensembles using non-rigorous methods. Aim 3: Extend rigorous re-weighting approach to protein loops. Aim 4: Create and maintain website. The proposed research will give valuable insight into the structure and function of protein loops and IDPs. The resulting software will provide us with a means to distinguish the biologically relevant structures from those that are not relevant. PUBLIC HEALTH RELEVANCE: The proposed research project will give insight into protein structure and function. Thus, our understanding of diseases caused by protein dysfunction will be enhanced.

DESCRIPTION (provided by applicant): The 4th Annual Symposium of the Intrinsically Disordered Proteins (IDP) Subgroup of the Biophysical Society will be held February 20, 2010, at the San Francisco Convention Center, San Francisco, California. The theme of the Symposium is Regulation and Utilization of Protein Disorder In Vivo. The program Co-Chairs, Elisar Barbar (Oregon State University) and Huang-Xiang Zhou (Florida State University) have developed an outstanding program, which includes nine invited speakers from the US, Canada, England, and Israel. Speakers include senior leaders in the field of IDPs as well as junior scientists who are having high impact in the field. In addition, two postdoctoral research awardees will be selected to speak at the Symposium. The traditional view of protein structure-function relationships posits a well-defined three-dimensional (3D) structure is required for function. However, it is now well appreciated that many biological functions are performed by highly dynamic proteins or protein domains that, in isolation, lack secondary and/or tertiary structure under physiological conditions. These proteins, termed intrinsically disordered proteins (IDPs), exist in organisms from all kingdoms of life and are most prevalent in eukaryotes. In mammals, IDPs mediate diverse cellular processes, including motility, metabolism and biosynthesis, division, and gene transcription. A point of special relevance to NIH is that IDPs are overrepresented in association with numerous human diseases such as cancer and neurodegenerative diseases. The tools traditionally used for determination of rigid protein structures are generally unsuitable in studies of IDPs due to their highly dynamic and disordered nature. However, NMR techniques which allow studies of dynamic protein ensembles have been adapted for studies of IDPs, as have other techniques such as small- angle X-ray scattering and single-molecule techniques. In addition, a wide variety of computational methods have emerged as powerful tools in studies of IDPs. The IDP Subgroup Annual Symposium has emerged as a leading forum for discussion of methodologies best suited for detailed studies of IDPs. The pace of studies of disordered proteins has been slow relative to output from projects such as the Protein Structure Initiative in the US, which is focused on highly ordered (folded) proteins. This has created a knowledge gap with respect to relationships between the structural and dynamic properties of thousands of IDPs (in humans) and the mechanisms that mediate their biological functions. Through its Annual Symposium, the IDP Subgroup seeks to develop and broaden awareness of new knowledge related to IDPs and new techniques for the characterization of their structure, dynamics, and biological functions. Support for this Symposium will reduce the knowledge gap noted above and promote everyone's long-range goal of improving treatment options for patients with cancer and other IDP-associated diseases. A major goal associated with this request for R13 funding is to provide support for young scientists to participate in the IDP Subgroup Annual Symposium, including two graduate students and four postdoctoral researchers. The IDP Subgroup and the Biophysical Society encourage participation of women, racial/ethnic minorities, persons with disabilities, and other individuals who traditionally have been underrepresented in science. PUBLIC HEALTH RELEVANCE: IDPs are involved in the pathogenesis of many human diseases, including cancer and neurodegenerative diseases. Therefore, expanding our knowledge of the structural features and functional mechanisms of IDPs through support of the IDP Subgroup Annual Symposium will provide insights into diverse biological processes. Furthering studies of IDPs will also drive new discoveries regarding the molecular mechanisms associated with devastating human diseases and provide new directions for therapeutics to combat these diseases.

? DESCRIPTION (provided by applicant): Intrinsic disorder controls the function of p53 and other cancer-associated IDPs PI's Daughdrill/Chen Project Summary/Abstract -- p53 is a tumor suppressor and cell cycle regulator that is activated by protein-protein interactions and posttranslational modifications (PTMs). Deletion or mutation of p53 can dramatically increase susceptibility to cancer. p53 is also an intrinsically disordered protein (IDP). IDPs are highly dynamic, do not form stable tertiary structures, and contain variable amounts of transient secondary structure. IDP domains are hotspots for PTMs and they frequently mediate protein-protein interactions through coupled folding and binding. IDP domains that interact with other proteins can contain defined levels of transient secondary structure that resemble their complex-bound structure. These levels of residual structure can modulate binding affinities with other proteins by tuning the change in conformational entropy that occurs during the coupled folding and binding reaction. Our recent publication in Nature Chemical Biology showed that levels of residual helicity in the disordered p53 transcriptional activation domain (p53TAD) controlled the binding affinity to the E3 ubiquitin ligase Mdm2, both in vitro and inside living cells. The levelsof residual helicity in free p53TAD were controlled by conserved prolines flanking the Mdm2 binding site. Mutating these prolines to alanine resulted in higher p53TAD helicity and stronger Mdm2 binding. This stronger Mdm2 binding abrogates the effects of PTMs leading to more rapid degradation of p53 following DNA damage. Lower levels of p53 reduce target gene expression and prevent cell cycle arrest. Our results suggest that precise levels of intrinsic disorder and residual helicity are necessary for regulating the p53-signaling network and changing the levels of disorder can modify the effects of phosphorylation and other PTMs. Studies from other groups have shown that PTMs can change intrinsic levels of disorder. Together levels of intrinsic disorder and PTM status allow IDP domains to dynamically respond to signaling changes in cellular networks. We propose to change the levels of intrinsic disorder in p53 and determine the effects on activation dynamics and target gene expression. We will also determine how intrinsic disorder combines with PTMs to control protein-protein interactions. Finally, we will investigate how the levels of intrinsic disorder in other cancer-associated IDPs control structure and function. The following specific aims are designed to accomplish these goals: Aim 1) Determine how intrinsic disorder controls the function of p53, Aim 2) Determine how intrinsic disorder combines with PTMs to control protein-protein interactions, and Aim 3) Determine how intrinsic disorder controls binding affinity and binding kinetics. To test these aims we will monitor the activation dynamics and target gene expression of p53 mutants using single-cell fluorescence microscopy, qPCR arrays, and reporter assays. To investigate how intrinsic disorder combines with PTMs to control protein-protein interactions and how intrinsic disorder controls binding affinity and binding kinetics we will primarily use NMR spectroscopy, isothermal titration calorimetry, and stopped-flow kinetics.

Regulation of transcriptional activation by protein disorder PIs: Daughdrill/Chen Abstract The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. It is mutated in at least 50% of solid tumors and some of these mutants are oncogenic and activate the transcription of genes that promote cell survival and metastasis. Despite decades of research on p53, there are currently no FDA approved drugs in the clinic that directly target wt or mutant p53. This is, in part, because 50% of p53 is disordered and we have an incomplete understanding of what these regions look like and how they function. We have recently shown that the disordered N-terminal acidic transactivation domain of p53 (NT) dynamically interacts with the DNA binding domain (DBD) with residues that contact DNA. This interaction inhibits DNA binding but increases binding specificity using a combination of nucleic acid mimicry and electrostatic shielding that enhances recognition of promoter binding sites in vivo. We propose to: (1) Determine the specific effects of nucleic acid mimicry and electrostatic shielding on DNA binding specificity, and how tethering controls the intramolecular interaction between NT and DBD. (2) Investigate how p53 NT phosphorylation regulates DNA binding affinity and specificity to create adaptable switching of transcriptional activation. (3) Determine how MdmX interferes with p53 DNA binding when it forms a heterodimer with p53. Successful completion of these experiments will lead to a better understanding of how p53 governs cell fate after DNA damage by regulating the binding specificity to pro-survival versus pro-apoptotic target genes. A deeper understanding of the structural and functional properties of the weak dynamic interactions within p53 and between p53 and MdmX is necessary for the successful development of small molecules to target these interactions for cancer therapy.