Ongoing advances in the elucidation of protein structures are leading to the production of large volumes of high-resolution data, in increasingly native environments. However, especially in the case of transmembrane proteins, experimental options for high-resolution functional analyses remain limited, and thus the usefulness of the structures in understanding human neurological diseases likewise remains limited. Moreover, the structures that are obtained represent single, static snapshots of proteins, although these likely have multiple conformations of functional significance. Thus, there is a growing need among the ion channel and membrane biophysics community, which is supported by the NINDS, for reagents that directly report on the functionality and dynamics of membrane proteins in live cells. An elegant solution to these problems is nonsense suppression, a method that makes it possible to encode any type of synthetic amino acid at a site of interest within a protein. These so-called unnatural amino acids can take the form of residues with single atom substitutions, or side-chains with novel fluorescent properties. Although multiple experimental avenues for the encoding of unnatural amino acids exist, each is associated with significant technical challenges. As such, this powerful approach remains inaccessible to most investigators studying molecular neuroscience. In 2014, in response to numerous inquiries by other investigators, the Ahern lab at the University of Iowa (UI) set out to simplify the dissemination of acylated orthogonal tRNAs, key components in nonsense suppression, for experiments involving eukaryotic membrane proteins. To this end, we made improvements to the underlying chemistry, making it more robust and allowing for the encoding of a more chemically diverse set of amino acids. In addition, these new reagents display vastly improved stability profiles thus allowing for easy shipping to laboratories throughout the U.S. With our reagents and guidance, a number of new user groups have successfully applied this previously difficult approach to a variety of membrane proteins relevant to the NINDS mission. Overall, these efforts have produced a high-functioning collaborative outreach service, ?The Facility for Atomic Mutagenesis.? This resource will provide broad access to custom reagents for nonsense suppression, and this facility is able to quickly adapt or design synthetic approaches to meet the interests of an application by new users. As the technologies become more standardized and our user group expands, we will scale accordingly, taking advantage of infrastructure present with the UI Carver College of Medicine and local industrial partners such as Integrated DNA Technologies. These collaborations will ultimately support more efficient dissemination of these research tools, to answer diverse questions in molecular neuroscience. Their use will be buoyed by a growing user base, annual training seminars, web-based forums and published protocols in open-access peer-reviewed journals. Our Advisory Board ? Kossiakoff, Perozo, Koide, Nakamoto ? will ensure efficient stewardship of key resources and the alignment of our strategic vision to NINDS.

Public Health Relevance

The proposed efforts will result in the efficient dissemination of reagents that will enable end users to perform atomic mutagenesis and to encode next generation powerful optical probes to advance the understanding of membrane proteins. Success of the proposed endeavors will enable the generation of powerful and widely applicable research tools and functional data that will be widely available to the NINDS research community.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Resource-Related Research Projects (R24)
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Special Emphasis Panel (ZNS1)
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Silberberg, Shai D
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University of Iowa
Schools of Medicine
Iowa City
United States
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Infield, Daniel T; Matulef, Kimberly; Galpin, Jason D et al. (2018) Main-chain mutagenesis reveals intrahelical coupling in an ion channel voltage-sensor. Nat Commun 9:5055
Infield, Daniel T; Lee, Elizabeth E L; Galpin, Jason D et al. (2018) Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape. J Gen Physiol 150:1017-1024
Infield, Daniel T; Lueck, John D; Galpin, Jason D et al. (2018) Orthogonality of Pyrrolysine tRNA in the Xenopus oocyte. Sci Rep 8:5166
Rivera-Acevedo, Ricardo E; Pless, Stephan A; Schwarz, Stephan K W et al. (2012) Extracellular quaternary ammonium blockade of transient receptor potential vanilloid subtype 1 channels expressed in Xenopus laevis oocytes. Mol Pharmacol 82:1129-35