Our long-term goal is to advance biomedical science by uncovering the mechanisms of proteins, such as K+ channels that underlie numerous physiologically and pathologically important processes including neuronal activity, blood pressure regulation and cardiac rhythmicity (or arrhythmias). Knowledge of protein mechanisms constitutes the necessary foundation for us to understand physiological and pathophysiological processes, and devise therapeutic strategies to treat diseases. The goal of this proposal is to develop a visible light microscope and use it to quantitatively examine angstrom-scale conformational changes of both soluble and membrane proteins on a physiologically relevant time scale, and to use this technique to uncover, for example, the control mechanisms of Ca2+-dependent K+ channels. Crystallography has produced abundant useful protein structures for understanding protein function. However, full understanding of a protein molecule must include both its spatial and temporal characteristics. We thus need to go beyond describing a protein merely with static pictures and, instead, represent it with a real-time motion picture, i.e., a digital model that simultaneously exhibits both its structural and its kinetic mechanisms with embedded energetic information. However, the required experimental information about protein dynamics is often lacking, due to the absence of relatively general methods for reliably tracking angstrom-scale conformational changes of a protein. Generally, such small changes can be reliably and quantitatively resolved only with such structural techniques as crystallography or Cryo-EM, which, unfortunately, lack time resolution. Conventional light microscopy, on the other hand, may be time-resolved but its spatial resolution remains too low to resolve angstrom-scale changes, despite the landmark achievement with ~20 nm resolution of super-resolution fluorescence microscopy. In the spirit of innovation, we will build a visible-light microscope?with angstrom- and millisecond-scale resolution?to examine multi-state protein-conformational changes in a single protein molecule. We will first develop it using the isolated, soluble control domain of Ca2+-dependent K+ channels as a model, and then extend the technique to membrane proteins, using intact channels. Additionally, in these studies, we will also systematically investigate the energetics and kinetics of the control (or ligand-gating) domain in these channels, information necessary for building a quantitative, mechanistic model that accounts of Ca2+- dependent gating-conformational changes. Such mechanistic inquiry cannot be carried out with existing electrophysiological and microscopic techniques. Success of our proposed study will transform the way we investigate the dynamics/kinetics of protein conformational changes, and accelerate the transition from the current, mostly static approach of structural biology to dynamic structural biology.

Public Health Relevance

The proposed studies are designed to advance visible light microscopy so that we can quantitatively monitor angstrom- and millisecond-scale conformational changes of proteins. This technique will be used in conjunction with electrophysiology to uncover the control mechanisms of K+ channels, a class of proteins that underlie a large set of physiologically and pathologically important processes including neuron activity, blood pressure regulation, and cardiac rhythmicity or arrhythmia.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055560-20
Application #
9597583
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Nie, Zhongzhen
Project Start
1997-05-01
Project End
2020-11-30
Budget Start
2018-12-01
Budget End
2019-11-30
Support Year
20
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Physiology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Pau, Victor; Zhou, Yufeng; Ramu, Yajamana et al. (2017) Crystal structure of an inactivated mutant mammalian voltage-gated K+ channel. Nat Struct Mol Biol 24:857-865
Combs, David J; Lu, Zhe (2015) Sphingomyelinase D inhibits store-operated Ca2+ entry in T lymphocytes by suppressing ORAI current. J Gen Physiol 146:161-72
Ramu, Yajamana; Xu, Yanping; Shin, Hyeon-Gyu et al. (2014) Counteracting suppression of CFTR and voltage-gated K+ channels by a bacterial pathogenic factor with the natural product tannic acid. Elife 3:e03683
Xu, Yanping; Ramu, Yajamana; Shin, Hyeon-Gyu et al. (2013) Energetic role of the paddle motif in voltage gating of Shaker K(+) channels. Nat Struct Mol Biol 20:574-81
Combs, David J; Shin, Hyeon-Gyu; Xu, Yanping et al. (2013) Tuning voltage-gated channel activity and cellular excitability with a sphingomyelinase. J Gen Physiol 142:367-80
Xu, Yanping; Ramu, Yajamana; Lu, Zhe (2010) A shaker K+ channel with a miniature engineered voltage sensor. Cell 142:580-9
Xu, Yanping; Shin, Hyeon-Gyu; Sz├ęp, Szilvia et al. (2009) Physical determinants of strong voltage sensitivity of K(+) channel block. Nat Struct Mol Biol 16:1252-8