248 research outputs found

    Genetic Incorporation of Unnatural Amino Acids for the Study of Protein-Protein Interactions.

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    Proteins comprise the majority of the cell and are vital to all cellular functioning. Protein-protein interactions (PPIs) are the communication networks behind cellular processes, often functioning in machine-like complexes with exchangeable subunits or parts to convey different messages. PPIs exhibit a wide range of structural features, surface areas, and affinities with some displaying dynamic interfaces allowing multiple binding partners to interact depending on cellular conditions. This makes some PPIs more difficult to study than others. Understanding these PPIs and exploring larger PPI networks has been a challenge without considering the cellular context in which they belong. Methods to study difficult PPIs in their native environments have thus been instrumental advancing the field. The predominant theme of this work is the demonstration of the utility of genetically incorporated photolabile unnatural amino acids for the study of the difficult PPIs between transcriptional activator-coactivator complexes. Covalent chemical capture of protein binding partners in live cells is combined with mass spectrometry to discover novel PPIs and further expanded to include new ways to visualize direct PPIs on DNA. Caveats to the covalent capture method are also explored with an illustration of capture efficiencies of two common photolabile groups across various PPI binding affinities and surface areas. The work presented here displays a thorough examination of the use and application of chemical capture for the study of PPIs in a cellular context. The methods established within this work add to the foundation for the study of difficult PPIs and demonstrates the ability to understand new networks of low affinity, dynamic interactions. The presentation of novel binding partners for the well-studied transcriptional activator, Gal4, expands traditional beliefs on transcriptional activator participation in binding dynamic complexes as well as highlights the potential of these PPIs for later therapeutic points of intervention. In addition, the groundwork for guidelines on using covalent chemical capture in various PPIs was established which, when completed, will enable not only easier use but also hopefully lead to the ability to tailor selection of a photocrosslinker based on the specific PPIs under study.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135876/1/rpricer_1.pd

    When Home is a Living Hell: Vulnerable Women and Sexual Harassment in Housing

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    Low-income women experience a nightmarish victimization when they are sexually harassed by landlords in their homes, homes many are desperate to keep. The staggering lack of data on this issue means laws and courts have been slow to address this phenomenon. Although courts have relied primarily on a Title VII employment-based sexual harassment framework to address this issue, it does not go far enough in protecting women in their homes. The home and the workplace are inherently different and thus require a different legal approach to redress the issue. This comment examines sexual harassment in housing and why Texas women are particularly susceptible to it. This comment further advocates for courts to look to existing housing law and expand application of the Fair Housing Act’s disparate impact approach in addressing sexual harassment in housing to low-income women. Low- income women of color are the primary victims of sexual harassment in housing; this permits a gender and race-based disparate impact theory as another framework through which courts may craft a solution, though it too has its shortcomings. Additional measures, such as the expansion of federal law to cover sexual harassment in housing, and state-level policy changes that would penalize deviant landlords and property owners, would serve to empower and strengthen low- income Texas women without leaving entire subsections of the population behind. This comment further argues that holistic community-level action is not only desirable, but necessary to educate communities and provide low-income women increased access to the courts. Finally, the above suggested measures will do little to change the outlook for low-income women if states fail to address the underlying issue that makes the need for housing so desperate: the affordable housing crisis

    An analysis of selected agricultural factors in Tennessee, 1930-1940

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    This is an historical study of Agriculture in the State of Tennessee for the decade 1930 through 1940 designed to display the efficacy of: (1) changes in the agricultural labor force, and (2) changes in the agricultural population as independent variables when correlated with: (A) percent change in the number of farms, (B) percent change in the average number of acres per farm, (C) change in the value of all products, and (D) percentage changes in total tenancy. Industrialization, as against agriculture, has been injected into this quantitative analysis by two methods (as shall be apparent later) which would permit severely-qualified generalizations to be drawn from the data relating to the industrialization of an area to concomitant changes in its agriculture

    Sequence context and crosslinking mechanism affect the efficiency of in vivo capture of a protein–protein interaction

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    Protein–protein interactions (PPIs) are essential for implementing cellular processes and thus methods for the discovery and study of PPIs are highly desirable. An emerging method for capturing PPIs in their native cellular environment is in vivo covalent chemical capture, a method that uses nonsense suppression to site specifically incorporate photoactivable unnatural amino acids (UAAs) in living cells. However, in one study we found that this method did not capture a PPI for which there was abundant functional evidence, a complex formed between the transcriptional activator Gal4 and its repressor protein Gal80. Here we describe the factors that influence the success of covalent chemical capture and show that the innate reactivity of the two UAAs utilized, ( p‐ benzoylphenylalanine (pBpa) and p ‐azidophenylalanine (pAzpa)), plays a profound role in the capture of Gal80 by Gal4. Based upon these data, guidelines are outlined for the successful use of in vivo photo‐crosslinking to capture novel PPIs and to characterize the interfaces. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 391–397, 2014.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102672/1/bip22395.pd
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