Membrane proteins are of extreme importance for human health and 30% of the human proteome consist of membrane proteins, which are key players in important cellular processes controlling and mediating the interaction between cells, regulate transport in and out of the cells and are also the major players in bioenergy conversion including respiration; 40% of all current drugs being targeted to membrane proteins. However knowledge of their structure and function at the atomic level is sparse, as the structures for less 800 unique membrane proteins has been determined to date. Furthermore, most of these structures show only a static picture of the membrane protein, while their function in the cell is highly dynamic. This proposal aims to develop novel methods to determine ?molecular movies? of membrane proteins ?at work,? and specifically, to determine the dynamics of the catalytic cycle of the cytochrome oxidase and to study the conformational dynamics of the beta-adrenergic G-protein coupled receptor. The proposal aims to develop and use a combination of methods that focus on time-resolved femtosecond (fs) X-ray crystallography with XFELs. Fs crystallography, pioneered by our team in the previous funding cycles, has revolutionized X-ray crystallography. It overcomes radiation damage, enabling structure analysis of biological macromolecules at room temperature under physiological conditions based on the new serial approach for structure determination, where tens of thousands of X-ray diffraction snapshots are collected from a stream of fully hydrated nano/microcrystals of proteins, interacting with fs X-ray pulses from a Free Electron Laser. New developments in nanocrystal growth and characterization, together with development of new injector technology and data evaluation methods, have progressed the new method of SFX at a very fast pace based on results from this project, which has led to 70 publications, 29 of them in high impact journals as well as patents (accepted and provisional applications filed). This R01 Renewal is based on the success of the previous work but explores new areas by shifting the major focus from the proof-of-concept to their improvement and application?towards molecular movies of the functional dynamics of two important membrane proteins: cytochrome c oxidase and the beta-adrenergic receptor.
In Aim 1 we will study the dynamics of the catalytic cycle of cytochrome c oxidase, a large-multi-protein membrane protein complex and the key enzyme in respiration, catalyzing the 4 electron 4 proton reduction of oxygen to 2 water molecules, whose detailed mechanisms is still a hot topic of debate.
Aim 2 is focused on the dynamics of the beta-adrenergic receptor and its complexes with arrestin and G-protein. In this Aim, we will combine new SFX method developments (including making the GPCR triggered by light to allow for very fast dynamics to be detected) with the time resolved cryo-EM studies of the GPCR complexes that will allow us to unravel motions on a time scale of seconds.

Public Health Relevance

Membrane proteins have an overwhelming impact on human health, with over 30% of human genes coding for membrane proteins and 40% of all drugs targeting these proteins. The aim of this proposal is to study the dynamics of membrane proteins, and specifically, cytochrome c oxidase, a large-multi-protein membrane protein complex and the key enzyme in respiration, and of the G-protein coupled receptor, beta-adrenergic receptor, and its complexes with arrestin and G-protein. To enable time-resolved studies of these proteins, we will continue to develop and apply the new method of serial femtosecond nanocrystallography (SFX) to the determination of the structure and dynamics of membrane proteins?a technology that is based on diffraction snapshots collected from a stream of nanocrystals using ultra-short femtosecond X-ray pulses that are provided by hard-X-ray lasers, delivering pulses so brief that they terminate before radiation damage occurs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM095583-10
Application #
10110015
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
2010-09-30
Project End
2024-01-31
Budget Start
2021-02-01
Budget End
2022-01-31
Support Year
10
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Arizona State University-Tempe Campus
Department
Other Basic Sciences
Type
Schools of Arts and Sciences
DUNS #
943360412
City
Tempe
State
AZ
Country
United States
Zip Code
85287
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Chapman, Henry N; Fromme, Petra (2017) Structure determination based on continuous diffraction from macromolecular crystals. Curr Opin Struct Biol 45:170-177
Stagno, J R; Liu, Y; Bhandari, Y R et al. (2017) Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography. Nature 541:242-246
Gati, Cornelius; Oberthuer, Dominik; Yefanov, Oleksandr et al. (2017) Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser. Proc Natl Acad Sci U S A 114:2247-2252
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Reddy, Hemanth K N; Yoon, Chun Hong; Aquila, Andrew et al. (2017) Coherent soft X-ray diffraction imaging of coliphage PR772 at the Linac coherent light source. Sci Data 4:170079
Ishigami, Izumi; Zatsepin, Nadia A; Hikita, Masahide et al. (2017) Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature. Proc Natl Acad Sci U S A 114:8011-8016
Zhou, X Edward; Gao, Xiang; Barty, Anton et al. (2016) X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex. Sci Data 3:160021
Coe, Jesse; Fromme, Petra (2016) Serial femtosecond crystallography opens new avenues for Structural Biology. Protein Pept Lett 23:255-72

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